1. Introduction
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), where autoreactive immune responses are involved in demyelination and CNS damage. The etiology and pathogenesis of the disease remain elusive. However, several lines of evidence demonstrate that adaptive immune response plays a key role in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), the mouse model of MS [
1,
2,
3]. The major components of the adaptive immunity, T cells, are initially activated by antigen presenting cells (APCs) in lymph nodes. Activated T cells migrate into the CNS across the blood brain barrier (BBB) and reactivated again in perivascular space, where CNS-resident cells including microglia and macrophages present myelin antigens to T cells [
4]. Thus, those activated CD4
+ T cells orchestrate the functions of other adaptive immune cells, such as CD8
+ T cells and B cells, as well as innate immune cells in the CNS and periphery [
5]. Depending on the composition of the cytokine milieu, naïve CD4
+ T cells may differentiate into different T helper (Th) subsets including Th1, Th2 and Th17 that produce signature cytokines such as IFNγ, IL-4 and IL-17 respectively. The differentiation of naïve CD4
+ T cells into Th1, Th2, or Th17 types are governed by transcription factors, known as Tbet, GATA3 and RORγt respectively [
6]. Th subsets affect the CNS inflammation in different ways. Th1 and Th17 responses potentiate the CNS inflammation, while Th2 response dampens inflammatory response and protects CNS damage [
6]. These findings highlight the importance of T cell-mediated immunity in MS pathology.
APCs including dendritic cells, macrophages and microglia are innate immune cells that trigger T cell activation [
7]. These cells create the first line of response by recognizing pathogens and/or danger signals via pattern-recognition receptors (PRR) [
8]. NOD-like receptors (NLRs) are intracellular PRR that are mainly expressed by cells of hematopoietic origin and regulate both innate and adaptive immune responses [
9]. Recently, NLRs have gained more attention since 3 members of the family including CIITA, Nlrc5 and Nlrp3 regulate transcription of molecules that shape adaptive immune responses. CIITA [
10] and Nlrc5 [
11,
12] show transcriptional activities for MHC II and MHC I molecules respectively, while
Nlrp3 acts as a Th2 transcription factor and promotes IL-4 production [
13]. In addition, activation of NLRs often leads to the production and secretion of pro–inflammatory cytokines such as IL-1β and IL-18 that in turn potentiate differentiation of Th1 and Th17 subsets [
9,
14]. These findings highlight the key role of NLR proteins in shaping T cell response and adaptive immunity.
Not all NLRs are pro–inflammatory.
Nlrp12 is a recently discovered member of NLRs that is shown to be a negative regulator of both canonical and non-canonical nuclear factor-κB (NF-κB) signaling pathways [
15]. Previous studies showed that Nlrp12
−/− mice are highly vulnerable to inflammatory diseases such as experimental colitis and colorectal tumor development [
16,
17,
18,
19]. In the context of CNS inflammation, the lack of
Nlrp12 resulted in increased CNS inflammation and exacerbated course of EAE [
19].
Nlrp12−/− mice developed earlier and more severe form of EAE than wild-type (WT) mice. This phenotype parallel with significant increases in the expression of pro-inflammatory genes in the spinal cords of
Nlrp12−/− mice relative to WT mice. Experiments using mouse primary microglia cultures demonstrated that
Nlrp12 significantly inhibits production of the inflammatory mediators such as
inducible nitric oxide synthase (iNOS), Tumor Necrosis Factor (TNF)α, IL-6 and nitric oxide (NO) [
19]. However, the ability of
Nlrp12 to modulate T cell responses remains poorly defined.
A recent article by Lukens et al. revealed that
Nlrp12 is expressed not only by myeloid cells but also by T cells. It negatively regulates NF-κB signaling, T cells proliferation and the secretion of Th1/Th2/Th17 cytokines [
20]. Non-surprisingly,
Nlrp12 deficient mice developed enhanced inflammatory symptoms in T-cell-mediated autoimmune diseases such as colitis and atopic dermatitis [
20]. However, in EAE model, lack of
Nlrp12 promotes Th2 response and IL-4 secretion, which results in a milder form of EAE with atypical symptoms, including ataxia and impaired balance control [
20]. Collectively, current findings and controversies indicate that the exact immunoregulatory functions of
Nlrp12 in T cell activation and T cell-mediated autoimmunity are poorly understood.
In this study, we investigated the immunoregulatory role of Nlrp12 in T cell responses using classical induced-EAE and spontaneous EAE (spEAE) models. We further characterized the role of Nlrp12 in regulating T cell receptor (TCR) signaling pathways and IL-2 production.
2. Materials and methods
2.1. Mice
All the protocols and procedures were approved by the University of Sherbrooke Animal Facility and Use Committee (Protocols #280-15, 4 April 2017; #335-17B, 22 February 2018).
Nlrp12 knock-out
(Nlrp12−/−) mice on C57BL/6J background were kindly provided by Dr. Jenny P.Y. Ting (Chapel Hill, NC, USA). Mice were backcrossed for at least 15 generation. The 2D2 transgenic mice expressing a TCR specific for the myelin oligodendrocyte (MOG
35–55) peptide were purchased from Jackson Laboratory.
Nlrp12−/− and WT mice were crossed with 2D2 mice to generate
Nlrp12−/− 2D2 mice. We genotyped all the animals for
Nlrp12 and 2D2 (Supplementary protocol) and only those animals that were
Nlrp12−/− and 2D2
+ were included in the study (
Supplementary Figure S1). Moreover, the expression of Vβ11 receptor was verified with flow cytometry. The mice were maintained under specific pathogen-free conditions in the animal facility of the faculty of medicine, at the University of Sherbrooke.
2.2. Induction of EAE and Tissue Collection
EAE was induced in 8–10-week old WT or
Nlrp12−/− female mice as previously described [
19]. An emulsion mixture of MOG
35−55 (Genemed Synthesis Inc., San Antonio, TX, USA), complete Freund’s Adjuvant (CFA) (Sigma-Aldrich, St. Louis, MO, USA) and
Mycobacterium tuberculosis H37 RA (Difco Laboratories, Detroit, MI, USA) was prepared and injected subcutaneously in the flank with a total of 200 μg MOG
35–55 and 500 μg
Mycobacterium. Mice were also injected intraperitoneally on days 0 and 2 with 200 ng Pertussis toxin (List Biological Laboratories Inc., Campbell, CA, USA). After 3 weeks of immunization, mice were sacrificed, perfused with ice-cold phosphate-buffered saline (PBS) (Wisent, St. Bruno, QC, Canada) and the CNS tissues were collected.
2.3. Intracellular Staining and Flow Cytometry
CD4+ T cells were purified from lymph nodes and spleens using Mouse CD4+ T Cell Isolation Kit (eBioscience, San Diego, CA, USA) and activated with plate-bound anti-CD3 (eBioscience, clone:145-2C11, 1 μg/mL) and anti-CD28 (eBioscience, clone: 37.51, 2 μg/mL) antibodies for indicated times. T cell proliferation was assessed by Ki67 intranuclear staining following fixation and permeabilization in the Foxp3/Transcription Factor staining kit (eBioscience). For intracellular staining of cytokines, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 500 ng/mL, Sigma Chemical Co., St. Louis, MO, USA) and ionomycin (1 μg/mL, Calbiochem Corp., La Jolla, CA, USA) for 5 h at 37 °C in the presence of Brefeldin A (10 μg/mL, eBioscience). Cells were stained with anti-CD4-FITC antibody (eBioscience), fixed, permeabilized and then stained with anti-IFNγ-PE, anti-IL-4-PE, IL-17-PerCP-Cy5.5, Tbet-PE, or RORγt-PE antibody, as per the manufacturer’s instructions (eBioscience). Sample acquisition was performed with Beckman Coulter CytoFlex (Beckman Coulter, Brea, CA, USA) and data were analyzed using CytExpert 2 software (Beckman Coulter, Brea, CA, USA). Plots were prepared using CytExpert 2 and FlowJo (San Carlos, CA, USA) software.
2.4. Quantitative RT-PCR
RNA was extracted from CD4
+ T cells using TRIzol reagent (Life Technologies Inc., Burlington, ON, USA) and cDNA was synthesized as previously described [
19]. Reverse transcription PCR (RT-PCR) was used to verify the expression of
Nlrp12 in activated T cell using KiCqStart™ SYBR
® Green qPCR ReadyMix (Sigma Aldrich, St. Louis, MO, USA). Primers (IDT, Coralville, IA, USA) sequences were as follows:
Nlrp12F: 5′-CCT CTT TGA GCC AGA CGA AG-3′,
Nlrp12R: 5′-GCC CAG TCC AAC ATC ACT TT-3′,
18SF: 5′-CGG CTA CCA CAT CCA AGG AA-3′ and
18SR: 5′-GCT GGA ATT ACC GCG GCT-3′. The relative expression was calculated using the ΔΔC
T method [
21].
2.5. Cytokine Measurement
IFNγ and IL-4 cytokines in the supernatant of activated CD4
+ T cell culture and tissue lysates were measured using ELISA kits as previously described [
19]. Briefly, lymph node, spinal cord and cerebellum tissues were homogenized in lysis buffer supplemented with protease inhibitors (Cell Signaling Technology, Danvers, MA, USA) by rapid agitation using 3-mm stainless beads and a TissueLyser II (Qiagen, Hilden, Germany) homogenizer for 2 min. The levels of IL-4 in tissue lysates were determined using a high sensitivity IL-4 ELISA Kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instruction. The amount of IFNγ was determined using IFNγ kit purchased from PeproTech (Rocky Hill, NJ, USA).
2.6. Differentiation of 2D2 T Cells Toward Th1 or Th17 In Vitro
Naïve CD4+ T cells were purified from the spleens and lymph nodes of Nlrp12−/− 2D2 and WT 2D2 mice using MagniSort Naïve CD4 T Cell Enrichment Kit (eBiosciences). Purified CD4+ T cells were stimulated with MOG (50 µg/mL) in the presence of WT splenocytes at 1:1 ratio and Th1-, Th2- or Th17- polarizing condition (Th1: IL-12 (10 ng/mL), anti-IL-4 (10 μg/mL), IL-2 (10 ng/mL); Th2: IL-4 (10 μg/mL), hTGF-β1 (10 ng/mL), IL-2 (10 ng/mL) and Th17: anti-IL-12 (10 μg/mL), anti-IL-4 (10 μg/mL), anti-IFN-γ (10 μg/mL), mIL-6 (10 ng/mL), hTGF-β1 (10 ng/mL)). Recombinant cytokines and antibodies were purchased from Biolegend (San Diego, CA, USA) and eBioscience (San Diego, CA, USA), respectively. After 72 h of culture, cells were stained for MOG-TCR transgenic surface marker, Vβ11 and Th1- or Th17- associated markers (intracellular cytokine and transcription factor). The percentage of Th1 or Th17 cells were evaluated by Flow cytometry.
2.7. T Cell Activation and Signaling Pathways
To analyze p65 phosphorylation upon CD3 cross-linking, CD4+ T cells were purified from the lymph nodes of Nlrp12−/− and WT mice and incubated with 10 μg/mL anti-CD3 (eBioscience, San Diego, CA, USA) for 30 min on ice, followed by washing and re-suspending the cells in serum-free medium. T cells were incubated with 10 μg/mL cross-linking anti-mouse IgG antibody (R&D systems, Minneapolis, MN, USA) for 20 min at 37 °C. Akt phosphorylation was quantified after activation of CD4+ T cells with PMA/Iono for 10 min at 37 °C, while the phosphorylation of S6 was analyzed after 24 h activation of CD4+ T cells with plate-bound anti-CD3/CD28 antibodies. Following stimulation in a defined period of time, T cells were washed and lysed in the lysis buffer plus proteinase and phosphatase inhibitor (Cell Signaling Technology). Proteins were then separated by on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Transfers were blocked for 1h at room temperature with 5% nonfat milk in TBS/0.1% Tween 20 (TBST) and then incubated overnight at 4 °C in the primary antibodies (Cell Signaling Technology) diluted in TBST. The membranes were washed 3 times with TBST and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Cell Signaling Technology) diluted 1/1000 in TBST for 1 h at room temperature. The immunoblots were developed with Lumigen ECL ultra reagent (Lumigen, Southfield, MI, USA), imaged with ChemiDocTM (Bio-Rad, Hercules, CA, USA) and analyzed using Image Lab software (Bio-Rad).
2.8. Phosphoflow Cytometry
Phosphorylation of S6 ribosomal protein was confirmed by flow cytometry. Following 24 h stimulation of CD4+ T cells with plate-bound anti-CD3/CD28, cells were immediately fixed and permeabilized using Fix and Perm Kit (eBioscience). After washing with perm buffer (eBioscience) and blocking with 5% Fetal Bovine Serum, cells were incubated with Phospho-S6 antibody (Cell Signaling Technology) for 1h at room temperature, followed by washing with perm buffer and incubation with Alexa Fluor® 555 (Cell Signaling Technology) conjugated antibody. Samples were acquired by Beckman Coulter CytoFlex and data was analyzed using CytExpert 2 software (Beckman Coulter, Brea, CA, USA).
2.9. SpEAE and Histological Analysis
WT 2D2 or
Nlrp12−/− 2D2 mice were monitored for the development of spEAE. Animals were sacrificed after 4 months of monitoring or after the development of EAE at the peak score of 4. The immunized mice were sacrificed as described in
Section 2.2 and the spinal cords were removed and fixed in 4% formaldehyde for 24 h. The spinal cord tissues were embedded in paraffin and cut into 5-μm sections and stained with hematoxylin and eosin (H&E) stain and immunofluorescence for astrocyte marker (GFAP), microglia marker (Iba1) and myelin basic protein (MBP). All slides were scanned using a digital slide scanner NanoZoomer-XR C12000 (Hamamatsu Photonics, Hamamatsu, Japan) and viewed using NDPview2 software (Hamamatsu Photonics, Hamamatsu, Japan).
2.10. Analysis of CNS-Infiltrating Mononuclear Cells by Flow Cytometry
Dissected spinal cords were filtered through a 70 mm nylon sieve (BD Pharmingen, Becton Dickinson, Franklin Lakes, NJ, USA) with a syringe plunger to get a uniform tissue homogenate that was digested with collagenase D (2.5 mg/mL, Roche Diagnostics, Risch-Rotkreuz, Switzerland) and DNase I (1 mg/mL, Sigma-Aldrich) with agitation at 37 °C for 45 min. Mononuclear cells were isolated by percoll (Sigma-Aldrich) centrifugation, in which 300 µL percoll was mixed with 1 mL cell suspension overlaid with 700 µL Hank’s Balanced Salt Solution (HBSS). The samples were centrifuged at 12,000× g for 15 min without break. Myelin and cell debris were aspirated and the mononuclear pellet was re-suspended and washed with HBSS before staining for surface markers and analysis by flow cytometry.
2.11. Statistical Analysis
All statistical analyses were conducted using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA). Results were expressed as the mean ± standard deviation. Statistical differences between WT and Nlrp12−/− samples were assessed by Student’s t-test. Level of Nlrp12 expression was assessed by one-way ANOVA and cellular signaling by two-way ANOVA. The significance level was set at p < 0.05.
4. Discussion
Early reports showed the expression and anti-inflammatory function of
Nlrp12 in innate immune cells of myeloid origin such as DC and macrophages [
18,
26]. However, a very recent report by Lukens et al. revealed the expression of
Nlrp12 in T cells [
20]. The current study aimed to investigate the immunoregulatory function of
Nlrp12 in T cell-mediated immune response in EAE. Our results suggest that
Nlrp12 plays pivotal role in Th1/Th2 balance by inhibiting Th1 peripheral responses in the favor of Th2. We demonstrated that in - lymph nodes of
Nlrp12−/− mice, Th1 to Th2 ratio is increased compared to WT mice. This shift, in part, can be explained by significant increases in the production of IFNγ by
Nlrp12−/− T cells. Interestingly,
Nlrp12 does not play a role in the differentiation of naïve T cells but upregulates IL-2 production and proliferation of CD4
+ T cells. The effect of
Nlrp12 is associated with its increased expression and inhibition of major molecular pathways including Akt and NF-κB in activated T cells.
Previously, we published that
Nlrp12−/− mice develop more severe form of EAE than WT mice, which is associated with exacerbated spinal cord inflammation and increased activation of microglia in
Nlrp12−/− mice compared to WT mice [
19]. In the present study, we found an increased Th1 dominant response in lymph nodes of
Nlrp12−/− EAE mice, suggesting that
Nlrp12 suppresses Th1 activation in the periphery. Interestingly, we did not find any change in the levels of IFNγ and IL-4 in the CNS from
Nlrp12−/− EAE mice compared to WT EAE mice. However, the IFNγ/IL-4 ratio significantly decreased in the spinal cord of
Nlrp12−/− EAE mice compared to WT EAE mice, which is consistent with Lukens et al. observation of enhanced Th2 response and increased IL-4 production in the CNS of
Nlrp12−/− EAE mice [
20]. In contrast to Lukens’ study where
Nlrp12−/− mice developed atypical EAE signs, we found severe classical EAE signs in
Nlrp12−/− mice compared to WT mice [
19]. Several plausible explanations of these discrepancies were proposed in our recent review [
9]. One possibility might be related to the difference in MOG-adjuvant immunization protocols between various labs. In our report, WT and
Nlrp12−/− animals were immunized with a total dose of 200 µg MOG [
19], which is two-fold higher than the immunization dose used by Lukens et al. [
20]. Interestingly, a recent study showed that immunization with low or high MOG concentration can modify the patterns of inflammatory cytokines [
27]. Their study demonstrates that anti-inflammatory cytokines such as IL-10 and TGFβ significantly increase in the CNS of EAE animal immunized with 100 µg MOG compared to 300 µg MOG immunization. Therefore, it is possible that the severe EAE signs in
Nlrp12−/− mice in our study were driven by lower levels of IL-10 and TGFβ anti-inflammatory cytokines or by higher levels of other inflammatory cytokines such as Granulocyte-macrophage colony-stimulating factor (GM-CSF) in the CNS [
28,
29]. Another possible explanation for observing different EAE profiles is the difference in the environmental conditions and different knockout strategies. It was shown that some C57BL/6 colonies have acquired a missense mutation in the
Nlrp12 gene that can affect neutrophil responses [
30]. In another study, genetic ablation of
Nlrp12 was found to cause significant changes in microbiota [
17]. Nevertheless, these variabilities highlight the complex immunoregulatory nature of
Nlrp12 that warrants further investigation.
Given the important role of T cells in the pathogenesis of EAE, we further investigated whether
Nlrp12 controls T cell proliferation and activation in a T cell-intrinsic manner. We found a significant increase of IFNγ and IL-4 levels in the supernatant of
Nlrp12−/− compared to WT T cells. However, when we measured the levels of intracellular cytokines by flow cytometry, higher percentage of CD4
+ IFNγ
+ T cells were found in
Nlrp12−/− group, while the percentages of CD4
+ IL-4
+ T cells or CD4
+ IL-17
+ T cells did not change between both groups. We addressed this discrepancy with Ki67 staining and our flow cytometry results revealed that activated
Nlrp12-/- T cells proliferate significantly more than WT T cells, which explains why we found increased production of both IFNγ and IL-4 in the supernatant of activated
Nlrp12−/− T cells. Consistent with our findings, Lukens et al., observed the higher expression of activation markers, enhanced proliferation and elevated secretion of Th1/Th2/Th17 cytokines by
Nlrp12−/− T cells compared to WT T cells in vitro [
20]. Collectively, these results show that
Nlrp12 inhibits the activation of inflammatory T cell subsets including Th1 and Th17.
In the presence of polarizing cytokines, we found no difference between
Nlrp12−/− or WT T cells in the expression of Th1- or Th17- associated molecules, suggesting that
Nlrp12 does not affect the differentiation of naïve T cells to Th1 or Th17. In a recent study by Cai et al., purified CD4
+ T cells from
Nlrp12−/− or WT mice were activated with anti-CD3 and polarizing cytokines for 6 days. They showed that the differentiation of
Nlrp12−/− T cells to Th1 or Th17 cells were significantly lower than WT T cells. However, no difference was found between WT and
Nlrp12−/− T cells in Th2 differentiation [
31]. Taken together, it appears that
Nlrp12−/− T cells respond differently to the environmental stimuli, depending on the type of activating signals, incubation period and polarizing conditions.
Our results, in agreement with Lukens’ study [
20], showed that
Nlrp12 inhibits T cell activation and proliferation. We found that the expression of
Nlrp12 was significantly increased in T cells following activation by anti-CD3 or anti-CD3/CD28 antibodies. The increased level of
Nlrp12 expression remained high even after 48 h stimulation of T cells, suggesting that
Nlrp12 modulates T cell signaling pathways upon TCR stimulation.
Since multiple signaling pathways are involved in T cell activation and proliferation, we hypothesized that signaling pathways were inhibited by
Nlrp12 in activated T cells. It is well-established that TCR activation triggers calcineurin (a Ca
2+/calmodulin-dependent phosphatase), MAPK and NF-κB signaling pathways, leading to the activation of the nuclear factor of activated T cell (NFAT), AP1 and NF-κB transcription factors that initiate IL-2 transcription [
22]. In this regard, we evaluated the phosphorylation of p65 (NF-κB subunit) and Akt in activated CD4
+ T cells. Western blot results demonstrated a significant increase in the phosphorylation of p65 in activated
Nlrp12−/− T cells compared to WT T cells, highlighting the inhibitory effect of
Nlrp12 on NF-κB signaling pathway (
Figure 10). These results are in agreement with previous studies that show
Nlrp12 suppresses canonical and non-canonical NF-κB pathways [
16,
20,
32,
33].
The NF-κB signaling cascade interacts with several parallel pathways including the signaling cascades initiated by phosphatidylinositol 3-kinase (PI3K) and Akt. We found a significant increase of Akt(Ser 473) phosphorylation in
Nlrp12−/− T cells compared to WT T cells. These results are supported by a study that demonstrates
Nlrp12 negatively regulates Akt signaling pathway in affected tumor tissues in colitis model [
16]. Interestingly, Akt acts upstream of NF-κB, where the activation of PI3K/Akt signaling pathway leads to phosphorylation and degradation of IκB protein, resulting in nuclear translocation and transcriptional activation of NF-κB [
34]. Taken together, our results demonstrate that
Nlrp12 inhibits Akt signaling pathway, which, subsequently, affects downstream pathways including NF-κB pathway. Moreover, phosphorylated Akt blocks the activity of two G1-checkpoint inhibitors, p21 and p27, and promotes cell cycle progression [
35]. Collectively, our results suggest that
Nlrp12 controls T cell proliferation and cytokine production by inhibiting the activation of Akt, that, in turn, affects several downstream effectors (
Figure 10).
One of the signaling pathways downstream of Akt phosphorylation is mTOR pathway, which induces protein synthesis and cell growth by regulating ribosomal p70S6 kinase 1 (S6K1) and eukaryotic translation factor 4E-binding protein 1 (4EBP1). S6K1 phosphorylates and activates S6, a ribosomal subunit involved in initiating protein synthesis machinery. Using western blotting and flow cytometry, we found no difference between
Nlrp12−/− T cells and WT T cells in the level of S6 phosphorylation. Therefore, it is possible that
Nlrp12 regulates protein synthesis and cell growth via modulating the activity of 4EBP1 (
Figure 10). Further investigation is warranted to uncover the regulatory mechanism of
Nlrp12 on mTOR signaling pathway.
The results of this study and previous publications suggest that
Nlrp12 inhibits NF-κB and MAPK signaling pathways in activated T cells [
20,
36]. The transcription of IL-2 gene is regulated by NF-κB, MAPK and Ca
2+/calmodulin-dependent pathways [
37,
38]. Accordingly, we hypothesized that
Nlrp12 inhibits IL-2 production by activated CD4
+ T cells. Flow cytometry data revealed a higher percentage of CD4
+ IL-2
+ T cells in activated
Nlrp12−/− CD4
+ cells compared to WT CD4
+ T cells, suggesting that
Nlrp12 suppresses IL-2 production by activated T cells. Incubation with cyclosporin inhibited IL-2 production by both
Nlrp12−/− and WT T cells, indicating that lack of
Nlrp12 does not affect Ca
2+/calmodulin signaling pathway in T cells.
Our in vitro findings, together with results obtained from in vivo EAE model, support the idea that
Nlrp12 inhibits T cell responses. In 2D2 mice, due to the presence of many MOG-reactive T cells, about 4–14% of mice developed spEAE [
39,
40]. In 2D2 mice, the percentage of Vβ11
+ T cells in the spleen were the same between healthy and spEAE animals, showing that our affected and non-affected animals had similar percentage of MOG-reactive T cells. However, a high percentage of MOG-reactive T cells infiltrated to the spinal cord of WT 2D2 spEAE mice, which confirms the presence of autoreactive T cells in inflamed spinal cord. The healthy
Nlrp12−/− 2D2 animals have only a few Vβ11
+ T cells in the spinal cord and do not contain pathology. The fact that none of
Nlrp12−/− 2D2 mice develop disease suggests that
Nlrp12 does not inhibit the development of spEAE and even can serve as contributing factor in the pathology of EAE. Due to slow rate of breeding of
Nlrp12−/− 2D2 mice (unpublished observation), these conclusions are based on the observation of thirty
Nlrp12−/− 2D2 mice. Interestingly, previous studies report that in induced EAE,
Nlrp12 can prevent [
19] or promote [
20] CNS inflammation. To explain the observed controversy, we propose a dual immunoregulatory function for
Nlrp12, in which
Nlrp12 can act as an inflammatory or anti-inflammatory molecule, depending on the type and severity of immunological challenge. The hypothetical model of
Nlrp12 immunoregulation is shown in
Figure 11.
The bifunctional nature of
Nlrp12 has been previously reported in several studies [
16,
19,
20,
31,
36,
41,
42]. Early in vitro studies suggest that
Nlrp12 is an inflammatory NLR that interacts with ASC to form inflammasome [
43]. Recent reports also support this idea and show that
Nlrp12 activates inflammasome in
Yersinia Pestis and
Plasmodium infections [
42,
44]. Moreover, several behavioral outcomes are similar between
ASC−/− and
Nlrp12−/− mice [
45]. On the other hand, there are studies that classify
Nlrp12 as an anti-inflammatory molecule and the inhibitor of NF-κB signaling pathway [
16,
18,
32,
46]. In this regard,
Nlrp12−/− mice were shown to be highly susceptible to inflammatory diseases of intestine such as experimental colitis and colon cancer [
16,
18]. Taken together, these findings support the dual immunoregulatory nature of
Nlrp12, that may vary in a cell-specific or stimulus-specific manner.
In conclusion, the present study provides new insight into the immunoregulatory role of Nlrp12 in T cell-mediated CNS inflammation, triggered by different modes of immunological challenges (induced EAE and spEAE). We demonstrated for the first time that Nlrp12 inhibits early TCR signaling pathways. In this way, Nlrp12 plays critical roles in balancing T cell response to control overt activation and maintain cellular homeostasis. Indeed, the fine-tuned triggering of Nlrp12 by different immunological challenges determine whether the outcome of the challenge would be an inflammatory or anti-inflammatory response. Factors determining the immunoregulatory function of Nlrp12 remain to be determined.