Peli1 promotes microglia-mediated CNS inflammation by regulating Traf3 degradation

Abstract

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.

Figure 1. Peli1-KO mice are resistant to EAE induction

(a) Mean clinical scores of age- and sex-matched wild-type (WT) and Peli1-KO mice subjected for MOG_35-55-induced EAE (n=5/group). (b) H&E and Luxol Fast Blue (LFB) staining of spinal cord sections from MOG_35-55-immunized wild-type and Peli1-KO EAE mice for visualizing immune cell infiltration and demyelinization, respectively (arrows). Scale bar, 100 μm. (c,d) Flow cytometry analysis of immune cell infiltration into the CNS (brain and spinal cord) of MOG_35-55-immunized wild-type and Peli1-KO mice (n = 5, day 15 post-immunization). Data are presented as a representative plot (c) and summary graph (d). (e) Absolute number of Th1 and Th17 cells in the CNS (brain and spinal cord) and draining LNs of MOG_35-55-immunized wild-type and Peli1-KO mice quantified by flow cytometry (n = 4, day 15 post-immunization). Data are presented as summary graphs. (f) QPCR analysis to determine the relative mRNA expression level of genes encoding chemokines (left panel) and proinflammatory cytokines (right panel) in spinal cords of MOG_35-55-immunized wild-type and Peli1-KO mice (n = 4, day 15 post-immunization). Data were normalized to a reference gene, β-actin. *P<0.05 and **P<0.01.

Figure 2. Peli1 deficiency in radioresistant cells inhibits immune cell recruitment into the CNS and ameliorates EAE pathogenesis

(a) EAE induction of wild-type and Peli1-KO mice adoptively transferred with GFP⁺ wild-type BM cells. (b–e) Flow cytometry analysis of total CNS-infiltrating cells (GFP⁺, b,c) and CNS-infiltrating CD4 T cells (GFP⁺CD4⁺, d,e) of the MOG_35-55-immunized wild-type and Peli1-KO GFP-chimeric mice described in a, showing a representative plot (b,d) and a summary graph (c,e). (f) Flow cytometry analysis of GFP⁺CD4⁺ cell number in the spleen of MOG_35-55-immunized wild-type and Peli1-KO GFP-chimeric mice. (g) QPCR analysis of the indicated genes. *P<0.05 and **P<0.01.

Figure 3. Peli1-mediated microglial activation contributes to EAE pathogenesis

(a,b) QPCR analysis of relative mRNA expression for Peli family members in non-treated primary microglia (MG), astrocytes (AC), oligodendrocyte precursor cells (OPC), and neurons (a) or in non-treated (NT) and LPS-stimulated microglia (b). (c) QPCR analysis of Peli family member expression in FACS-sorted GPF^–CD11b⁺ microglia isolated from the non-immunized or EAE-induced (15 day after MOG_35-55 immunization) GFP-chimeric mice described in Fig. 2a. (d) Flow cytometry analysis of activation markers (CD45 and MHC class II) on gated GFP⁻CD11b⁺ microglia isolated from the CNS of wild-type or Peli1-KO GFP-chimeric mice, which were either non-immunized or immunized for the indicated days of EAE induction. Data are representative of 4 animals per group and time points. (e) Summary graph of d, showing the mean ± S.D. of the frequency value of resting (GFP⁻CD11b⁺CD45^loMHC II⁻) and activated (GFP⁻CD11b⁺CD45^hiMHC II⁺) microglia at the two indicated time points. (f) Flow cytometry analysis of MHC II expression on wild-type and Peli1-KO primary microglia that were either not treated (NT) or stimulated with LPS in vitro for the indicated times. (g) QPCR analysis of the indicated genes in FACS-sorted GPF^–CD11b⁺ microglia isolated from EAE-induced (day 15 post-immunization) wild-type or Peli1-KO GFP-chimeric mice. (h) EAE induction in wild-type or Peli1-KO mice that were stereotaxically injected, on day 3 of MOG_35-55 immunization, with PBS, Peli1-KO microglia (KO MG), or wild-type microglia (WT MG). Mean clinical scores were determined based on 5 mice per group. *P<0.05 and **P<0.01.

Figure 4. Peli1 mediates TLR-stimulated gene expression and MAPK activation in microglia

(a,b) QPCR analysis of relative mRNA expression for the indicated proinflammatory cytokine genes (a) and chemokine genes (b) in wild-type and Peli1-KO microglia stimulated with ligands of different TLRs: TLR4 (LPS, 100 ng ml^–), TLR9 (CpG, 2.5 μM^–), TLR7/8 (R837, 1 μg ml^–), TLR1/2 (Pam₃CSK₄, 1 μg ml^–), and TLR3 (poly(I:C), 10 μg ml^–). (c,d) IB analysis of phosphorylated (P-) and total MAPKs in whole-cell lysates of wild-type or Peli1-KO microglia stimulated with LPS (100 ng ml^–) (c) or Pam₃CSK₄ (1 μg ml^–) (d). The protein bands were quantified using ImageJ and presented as fold of phosphorylated over total MAPKs. Data are mean±S.D. values based on three independent experiments. *P<0.05 and **P<0.01.

Figure 5. Peli1 regulates TLR-stimulated c-IAP2 ubiquitination and Traf3 degradation

(a) Ubiquitination of Traf6 (upper) and c-IAP2 (lower) in LPS-stimulated wild-type or Peli1-KO microglia. (b) Ubiquitination of c-IAP2 (upper) and expression of the indicated proteins (lower) in HEK293 cells transfected with (+) or without (–) the indicated expression vectors. (c) Ubiquitination of c-IAP2 (upper) and expression of the indicated proteins (lower) in HEK293 cells transfected with (+) or without (–) the indicated cDNA expression vectors or Peli1 shRNA. (d) IB analysis of Traf3 and actin in whole-cell lysates of LPS-stimulated wild-type and Peli1-KO microglia. (e) Analysis of Traf3 K48 ubiquitination in wild-type or Peli1 KO microglia stimulated with LPS in the presence of a proteasome inhibitor, MG132. (f) IB analysis of Traf3 and actin in whole-cell lysates of FACS-sorted GPF^–CD11b⁺ microglia isolated from non-immunized (0 d) or EAE-induced (7 and 14 d) GFP-chimeric mice (as described in Fig. 2a). NS indicates a nonspecific band. (g) Ubiquitination of Traf3 (upper) and expression of the indicated proteins (lower) in HEK293 cells transfected with the indicated expression vectors. (h) Ubiquitination of Traf3 (upper) and expression of the indicated proteins (lower) in HEK293 cells transfected with (+) or without (–) the indicated expression vectors and subsequently (after 10 h of transfection) cultured in the absence (–) or presence (+) of Smac mimetic (SM, 1 μM) for another 24 h.

Figure 6. Traf3 ablation restores proinflammatory gene induction in Peli1-deficient microglia and EAE induction in Peli1-deficient mice

(a) QPCR analysis of the indicated mRNAs in nontreated (NT) or LPS-stimulated wild-type and Peli1-KO microglia infected with control vector or Traf3 shRNA. *P<0.05. (b) Mean clinical scores of the indicated mice subjected for MOG_35-55-induced EAE (n=5/group). (c,d) Flow cytometry analysis of immune cell infiltration into the CNS (brain and spinal cord) of the indicated mice at 30 days following MOG_35-55-mediated EAE induction (n = 4). Data are presented as a representative plot (c) and summary graph (d).