Peli1 promotes microglia-mediated CNS inflammation by regulating Traf3 degradation.

Microglia are crucial for the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Here we show that the E3 ubiquitin ligase Peli1 is abundantly expressed in microglia and promotes microglial activation during the course of EAE induction. Peli1 mediates the induction of chemokines and proinflammatory cytokines in microglia and thereby promotes recruitment of T cells into the central nervous system. The severity of EAE is reduced in Peli1-deficient mice despite their competent induction of inflammatory T cells in the peripheral lymphoid organs. Notably, Peli1 regulates Toll-like receptor (TLR) pathway signaling by promoting degradation of TNF receptor-associated factor 3 (Traf3), a potent inhibitor of mitogen-activated protein kinase (MAPK) activation and gene induction. Ablation of Traf3 restores microglial activation and CNS inflammation after the induction of EAE in Peli1-deficient mice. These findings establish Peli1 as a microglia-specific mediator of autoimmune neuroinflammation and suggest a previously unknown signaling mechanism of Peli1 function.

INTRODUCTION

Multiple sclerosis (MS) is an inflammatory disorder of the central nervous system (CNS), characterized by demyelination and axonal damage[1]. A widely used animal model for MS studies is experimental autoimmune encephalomyelitis (EAE)[2]. Initiation of EAE involves peripheral priming of myelin-specific autoimmune T helper 1 (Th1) and Th17 cells and their subsequent migration into the CNS, where they become reactivated by antigen-presenting cells (APCs) displaying myelin-derived peptides and engage in inflammatory processes. These pathological events culminate in disseminated CNS inflammation, leading to the destruction of oligodendrocytes and neurons, and the development of disease symptoms characterized by progressive paralysis[3]. The pathogenesis of EAE and MS also critically involves microglia[4-6], innate immune cells that seed the CNS early during embryonic development and differ in ontogeny from bone marrow (BM)-derived monocytes and macrophages[7,8]. Microglia sense microbial invasion and tissue damages, and upon activation, they secrete chemokines and proinflammatory cytokines, thereby mediating leukocyte recruitment into the CNS and the induction of inflammation[9,10]. Activated microglia also function as APCs in the reactivation of T cells within the CNS[9,11].

Microbial infections serve as an environmental trigger for the onset and persistence of MS[1,12] as well as the induction of EAE[13-15]. Consistently, receptors that detect pathogen-associated molecular patterns, such as toll-like receptors (TLRs), have an important role in the regulation of MS and EAE[16]. In particular, the TLRs expressed on microglia are thought to mediate microglial activation and CNS inflammation[16-19]. TLRs respond to both microbial components and endogenous damage-associated molecular patterns released from apoptotic cells or wounded tissues during the course of autoimmune inflammations. Upon stimulation by their ligands, TLRs elicit cascades of signaling events that lead to activation of the IκB kinase (IKK) and MAP kinases (MAPKs) as well as their downstream transcription factors, NF-κB, AP-1, and related DNA-binding proteins[20]. These transcription factors act cooperatively to induce the expression of a plethora of genes involved in leukocyte migration and inflammation[20].

The MyD88-dependent TLR pathway is particularly important for the pathogenesis of EAE[16,17]. An E3 ubiquitin ligase, c-IAP, is required for the activation of MAPKs, although not IKK, by the MyD88-dependent TLRs[21]. Upon activation by the TLR signals, c-IAP mediates K48 ubiquitination and degradation of Traf3, an action required for MAPK activation[21,22]. However, the mechanism mediating this c-IAP-Traf3 axis of TLR signaling is obscure. The Peli (also called Pellino) family of proteins has been implicated in the regulation of TLR and IL-1 receptor (IL-1R) signaling in innate immune cells[23-25]. Mammalian cells express three highly homologous Peli members, which complicates the study of their in vivo functions due to potential functional redundancies. Our recent study suggests that in innate immune cells and mouse embryonic fibroblasts (MEFs), Peli1 is dispensable for signal transduction by IL-1R and the MyD88-dependent TLRs, although Peli1 has a nonredundant role in mediating NF-κB activation by the TRIF-dependent TLR pathway[26]. Whether Peli1 is required for MyD88 TLR signaling in specific cell types and how Peli1 regulates TLR signaling in vivo in pathological processes remain elusive. In the present study, we discovered a crucial role for Peli1 in regulating microglial activation and EAE pathogenesis. We provide molecular and genetic evidence that Peli1 mediates the c-IAP-Traf3 signaling axis and, thereby, controls TLR-stimulated Traf3 degradation and MAPK activation.

RESULTS

Peli1-deficient mice are refractory to EAE induction

To study the role of Peli1 in CNS inflammation, we induced EAE in wild-type and Peli1-KO mice using a myelin oligodendrocyte glycoprotein (MOG) peptide (MOG35–55). As expected, wild-type mice developed severe clinical symptoms that were characterized by a gradual increase in the severity of paralysis (), inflammatory cell infiltration into the CNS (, left), and demyelination (, right). Despite the competent responses of the Peli1-KO T cells to antigen stimulation (), these mutant mice were highly resistant to EAE induction (). This phenotype of the Peli1-KO mice was associated with reduced frequency and number of CNS-infiltrating T cells (CD4+ and CD8+), myeloid cells and activated resident microglial cells (CD11b+CD45hi), and total lymphocytes (CD11b–CD45hi), with concomitant increase in resting microglial cells (CD11b+CD45lo) ().

The CNS of Peli1-KO mice also had a greatly reduced number of Th1 and Th17 cells, coupled with their accumulation in the draining lymph nodes (LNs) (). Notably, the CNS of EAE-induced Peli1-KO mice had defective expression of various chemokines and inflammatory cytokines known to mediate immune cell recruitment and inflammation (). These results suggest that the Peli1-KO mice may have impaired recruitment of T cells into the CNS.

Peli1 regulates EAE induction in radioresistant cells

To examine the cellular mechanism by which Peli1 regulates EAE pathogenesis, we performed passive EAE induction by adoptively transferring activated MOG-specific T cells into sublethally irradiated wild-type and Peli1-KO recipient mice. Transfer of wild-type T cells to wild-type recipient mice (WT to WT), but not to the Peli1-KO recipient mice (WT to KO), led to severe EAE disease, suggesting a CNS-specific role for Peli1 in mediating EAE pathogenesis (). On the other hand, Peli1 was dispensable for the EAE-inducing function of T cells, since the MOG-specific Peli1-KO T cells were fully competent in EAE induction in wild-type recipients ().

We next generated radiation BM chimeric mice by reconstituting lethally irradiated wild-type or Peli1-KO mice with wild-type BM cells isolated from a GFP-transgenic mouse[27]. The wild-type GFP-chimeric mice were highly susceptible, while the KO GFP-chimeric mice were refractory, to EAE induction (). A reverse BM transfer experiment revealed that Peli1 was dispensable in BM cells for mediating EAE pathogenesis (). These data further support a CNS-specific function for Peli1 in mediating EAE pathogenesis.

Taking advantage of the GFP-chimeric mice described above, we determined whether Peli1 functions in CNS-resident cells to mediate the recruitment of immune cells into the CNS during EAE induction. On day 5 following EAE induction, the CNS of both the wild-type and Peli1-KO chimeras had a low frequency of immune cells (GFP+) (). Over time, the frequency of the CNS-infiltrating immune cells was substantially increased in the wild-type chimeras, but not in the Peli1-KO chimeras (). Similarly, the Peli1-KO chimeras had a severe defect in recruiting CD4+ T cells into the CNS (). This was not due to a defect in donor T-cell activation, since the spleen of these mutant chimeras contained a considerably higher number of donor CD4+ T cells (). The CNS of Peli1-KO chimeric mice had considerably reduced expression of proinflammatory cytokine and chemokine genes (). Thus, Peli1 appears to function within the CNS-resident cells to mediate the induction of genes involved in immune cell recruitment and inflammation.

Peli1 has a microglia-specific role in EAE regulation

The brain and spinal cord were among the tissues with abundant Peli1 expression (). In particular, Peli1 was the predominant Peli family member expressed in microglia, whereas at least two of the three Peli family members were comparably expressed in astrocytes, oligodendrocyte precursor cells (OPCs), and neurons () or innate immune cells[28]. Furthermore, Peli1, but not the other Peli family members, was induced in microglia upon in vitro stimulation by LPS () or during in vivo EAE induction (). The Peli1 induction along with EAE was transient ().

To examine whether Peli1 is required for microglial activation during EAE induction, we took the advantage of the GFP-chimeric mice to distinguish between the infiltrating macrophages (CD11b+GFP+) and CNS-resident microglia (CD11b+GFP–). We detected microglial activation based on their well-defined induction of CD45 and MHC class II (MHC II)[5]. Following EAE induction, the wild-type chimeras substantially increased the frequency of activated microglia (CD45hiMHC II+GFP–) and decreased the frequency of resting microglia (CD45loMHC II–GFP–), whereas the Peli1-KO chimeras contained predominantly the resting microglia (). Purified Peli1-deficient microglia were also defective in LPS-stimulated MHC II expression in vitro (). The Peli1-deficient microglia, isolated from EAE-induced mice, also had impaired expression of proinflammatory cytokine and chemokine genes (). These results suggest the requirement of Peli1 for EAE-associated microglial activation.

As a more direct approach to examine the microglia-specific function of Peli1 in mediating EAE pathogenesis, we stereotaxically injected purified microglia () into the cerebrospinal fluid of Peli1-KO mice. Injection with the wild-type microglia, but not with the Peli1-KO microglia, rendered the Peli1-KO mice highly sensitive to EAE induction (). These data further emphasize a microglia-specific role for Peli1 in mediating CNS inflammation and EAE induction.

Peli1 mediates TLR-stimulated gene expression in microglia

Although microglia did not appreciably respond to the TLR3 ligand, poly(I:C), they responded to several ligands of MyD88-dependent TLRs, including LPS (TLR4), CpG (TLR9), R837 (TLR7), and Pam3CSK4 (TLR1/2), leading to induction of several proinflammatory cytokine and chemokine genes (). The Peli1 deficiency severely attenuated the gene induction by these MyD88-dependent TLR ligands ().

EAE pathogenesis also involves the CNS-resident astrocytes, which respond to proinflammatory cytokines, such as IL-17, IFN-γ, and TNF-α. However, the Peli1 deficiency did not appreciably affect such responses in astrocytes (). This result suggests a dispensable role for Peli1 in IL-17R signaling or functional redundancy of Peli1 with other Peli family members that are also expressed in astrocytes ().

Peli1 mediates TLR-stimulated MAPK activation in microglia

We have previously shown that in MEFs, Peli1 mediates NF-κB activation by the TRIF-dependent TLR pathway, but is dispensable for the MyD88-dependent TLR pathway[26]. Similarly, we found that the loss of Peli1 in microglia impaired the activation of NF-κB stimulated by the TLR3 ligand poly(I:C) (). In contrast, Peli1 was dispensable for NF-κB activation by the MyD88-dependent TLR ligand Pam3CSK4 (). The LPS-induced NF-κB activation in Peli1-deficient microglia was partially defective (), reflecting the fact that LPS stimulates both TRIF- and MyD88-dependent pathways[20]. These results further emphasize that Peli1 regulates NF-κB activation specifically in the TRIF-dependent TLR pathway.

The requirement of Peli1 in gene induction by MyD88-dependent TLRs in microglia () suggested a novel signaling function of Peli1. Indeed, the Peli1 deficiency severely attenuated the LPS-stimulated activation of three major families of MAPKs: ERK, JNK, and p38 (). This function of Peli1 appeared to involve the MyD88-dependent pathway, since Peli1 was also required for MAPK activation by Pam3CSK4 ().

Peli1 mediates TLR-stimulated c-IAP ubiquitination and TRAF3 degradation

Upon LPS stimulation, activated Traf6 undergoes autoubiquitination[23,24] and mediates MAPK activation by inducing K63 ubiquitination of c-IAP, a modification that triggers the ubiquitin ligase activity of c-IAP[21,22]. We found that the Peli1 deficiency did not inhibit Traf6 ubiquitination but severely attenuated c-IAP2 ubiquitination in LPS-stimulated microglia (). Since Traf6 is required for Peli1 activation[29], we surmised that Peli1 might mediate Traf6-induced c-IAP2 ubiquitination. We found that although both Traf6 and Peli1 induced K63 ubiquitination of c-IAP2, the wild-type Peli1 markedly promoted the Traf6-mediated c-IAP2 ubiquitination (). Conversely, the Traf6-induced c-IAP2 ubiquitination was blocked by expression of the Peli1 RING-deletion mutant (Peli1ΔC) () and by knocking down endogenous Peli1 by shRNA (). Moreover, the LPS-stimulated K63 ubiquitination of c-IAP2 was attenuated in the Peli1-deficient microglia (). These results suggest that Peli1 is essential for the induction of c-IAP ubiquitination by LPS and Traf6.

An important function of c-IAP in the TLR pathway is to mediate K48 ubiquitination and degradation of Traf3, thereby triggering MAPK activation[21,22]. We found that LPS stimulated rapid degradation of Traf3 in wild-type microglia (), although such a signaling event was much less prominent in BM-derived macrophages (data not shown). The LPS-stimulated Traf3 degradation was blocked in the Peli1-deficient microglia (), which could be rescued by reconstitution with exogenous Peli1 (). Consistently, LPS stimulated Traf3 K48-ubiquitination in wild-type microglia, which was diminished in the Peli1-deficient microglia (). A parallel experiment revealed that the TLR2 ligand Pam3CSK4 also induced Traf3 degradation in wild-type, but not Peli1-deficient, microglia (). Of note, Traf3 was gradually depleted in the microglia of wild-type mice but was accumulated in the microglia of Peli1-KO mice along with EAE induction (). Thus, Peli1 mediates the ubiquitin-dependent Traf3 degradation both in vitro upon TLR stimulation and in vivo along with EAE induction.

Consistent with a recent report that IKKi (also known as IKKε) activates Peli1[30], we found that overexpressed Peli1 induced Traf3 ubiquitination, which was promoted by IKKi (). Because c-IAP is known as an E3 of Traf3[21], we asked whether Peli1-induced Traf3 ubiquitination required c-IAP. We found that the Peli1-induced Traf3 ubiquitination was blocked in cells treated with the c-IAP inhibitor Smac mimetic[31-33] () or transfected with the c-IAP1,2 shRNAs (). Collectively, these results suggest that Peli1 activates c-IAP and participates in c-IAP-mediated Traf3 ubiquitination and degradation.

Traf3 ablation restores microglial activation and EAE sensitivity in Peli1-KO mice

To assess the functional significance of Traf3 degradation in mediating microglial activation, we knocked down Traf3 in Peli1-deficient microglia. Infection of the Peli1-KO microglia with a Traf3-specific shRNA, but not a control shRNA vector, largely rescued their defect in LPS-stimulated gene induction (). Consistent with this in vitro finding, the Traf3fl/flLysM-Cre mice, which had specific Traf3 ablation in microglia and other myeloid-lineage cells, were evidently more sensitive to EAE induction than the control Traf3+/+LysM-Cre mice (). This phenotype was not due to elevation in the production of Th1 or Th17 cells () or in the recall responses of MOG-specific T cells (). On the other hand, the microglia derived from the Traf3fl/flLysM-Cre mice were hyper-responsive to LPS-stimulated expression of proinflammatory cytokine and chemokine genes ().

The myeloid ablation of Traf3 also rescued the defect of the Peli1-KO mice in EAE induction () and in CNS-infiltration of T cells and CD45+CD11b+ myeloid cells (). Moreover, the Traf3 ablation rescued the defect of the Peli1-KO mice in microglial activation, as evidenced by the lack of the accumulation of resting microglial population (CD45loCD11b+) (). Taken together, these in vitro and in vivo studies highlight the functional significance of Peli1-mediated Traf3 degradation in mediating microglial activation and CNS inflammation.

DISCUSSION

Microglial activation is an early event in the development of CNS inflammatory disorders, and ablation or functional inhibition of microglia ameliorates EAE induction without affecting peripheral T-cell activation, emphasizing the promise of targeting microglial activation in the treatment of MS[4,34]. In the present study, we identified the E3 ubiquitin ligase Peli1 as a pivotal mediator of microglial activation and EAE pathogenesis. We have previously shown that peripheral innate immune cells and MEFs express different Peli family members at comparable levels[28], which suggests functional redundancies in some of their functions and explains why loss of Peli1 in peripheral innate immune cells selectively affects the TRIF-dependent TLR signaling in the NF-κB pathway[26]. Our present study revealed that microglia predominantly expressed Peli1 and required Peli1 for their activation during the course of EAE induction and TLR stimulation. In addition to attenuated TRIF-dependent NF-κB activation, the Peli1-deficient microglia had impaired activation of MAPKs stimulated by MyD88-dependent TLRs. Like microglia, T cells predominantly express Peli1; however, Peli1 is not required, but it rather serves as a negative regulator for T-cell activation[28]. In contrast to microglia, several other CNS cell types, including astrocytes, OPCs, and neurons, express different Peli family members at comparable levels. Although the role of Peli1 in mediating TLR signaling in these cell types needs to be further studied, the pattern of Peli expression predicts functional redundancy.

Microglia form a major population of CNS APCs that become activated along with EAE induction[10]. We found that Peli1 is crucial for mediating microglial activation both in vivo along with EAE induction and in vivo by LPS stimulation. These findings suggest the requirement of Peli1 in T-cell activation within the CNS. Peli1 is also required for the induction of proinflammatory cytokines and chemokines in microglia, which explains why the Peli1 deficiency impairs immune cell infiltration into the CNS and ameliorated EAE pathogenesis.

A major signaling function of Peli1 in microglia was to mediate the activation of MAPKs by MyD88-dependent TLRs. Peli1 deficiency did not interfere with the TLR-mediated activation of Traf6 but rather appeared to prevent Traf6 from mediating the K63 ubiquitination of c-IAP. Our data are in line with the recent report that Peli1 is a downstream target of Traf6 in the MyD88 TLR pathway[29]. The K63 ubiquitination of c-IAP is known to trigger its K48 ubiquitin ligase activity, allowing c-IAP to engage ubiquitin-dependent degradation of Traf3[21,22]. Consistently, Peli1 was required for Traf3 degradation in microglia both in vitro upon TLR stimulation and in vivo along with EAE induction. By different approaches, we demonstrated that the Traf3 accumulation in Peli1-deficient microglia contributes to the impaired inflammatory responses and ameliorated EAE in the Peli1-KO mice. Our data are in agreement with a previous report that Traf3 suppresses IL-17-stimulated signaling and EAE pathogenesis in radioresistant cells, likely the CNS cells[35]. Since IL-17 does not induce Traf3 degradation[35], it is likely that the Peli1 may not be directly involved in IL-17R signaling and that the Peli1-mediated Traf3 degradation in the TLR pathway may indirectly promote the IL-17R signaling.

It is currently unclear whether Peli1 is involved in human MS pathogenesis. Nevertheless, a recently published microarray data set suggests elevated Peli1 expression in the initial white matter lesion areas of MS brains[36] (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32915). We also performed microarray studies to analyze gene expression in the grey matters of several MS and non-MS brains. While Peli1 expression was largely comparable between the MS and non-MS samples, one MS sample showed a markedly higher level of Peli1 expression (). One technical issue was that the brain tissues contain only a small percentage of microglial cells. Nevertheless, these human study data generally suggest that the expression of Peli1 is altered in MS.

In summary, our work establishes Peli1 as a novel mediator of microglial activation and EAE pathogenesis. Based on our data, we propose a model that Peli1 functions downstream of Traf6 in the MyD88 TLR pathway to mediate the activation of c-IAP and induction of ubiquitin-dependent degradation of Traf3, thereby contributing to the activation of MAPKs and the induction of genes involved in microglial activation and CNS inflammation.

METHODS

Methods and any associated references are available in the online version of the paper.