Experimental Autoimmune Encephalomyelitis in the Mouse.

This article details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Additionally, active induction of EAE in the C57BL/6 strain using myelin oligodendrocyte glycoprotein (MOG) peptide is also discussed. Detailed materials and methods required for the purification of both PLP and MBP are described, and a protocol for isolating CNS-infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains. © 2021 Wiley Periodicals LLC. Basic Protocol: Active induction of EAE with PLP, MBP, and MOG protein or peptide Alternate Protocol: Adoptive induction of EAE with PLP-, MBP-, or MOG-specific lymphocytes Support Protocol 1: Purification of proteolipid protein Support Protocol 2: Purification of myelin basic protein Support Protocol 3: Isolation of CNS-infiltrating lymphocytes.

Cytokines and Chemokines in the Pathogenesis of Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis is a CD4+ T cell-mediated demyelinating disease of the CNS that serves as a model for multiple sclerosis. Cytokines and chemokines shape Th1 and Th17 effector responses as well as regulate migration of leukocytes to the CNS during disease. The CNS cellular infiltrate consists of Ag-specific and nonspecific CD4+ and CD8+ T cells, neutrophils, B cells, monocytes, macrophages, and dendritic cells. The mechanism of immune-mediated inflammation in experimental autoimmune encephalomyelitis has been extensively studied in an effort to develop therapeutic modalities for multiple sclerosis and, indeed, has provided insight in modern drug discovery. The present Brief Review highlights critical pathogenic aspects of cytokines and chemokines involved in generation of effector T cell responses and migration of inflammatory cells to the CNS. Select cytokines and chemokines are certainly important in the regulatory response, which involves T regulatory, B regulatory, and myeloid-derived suppressor cells. However, that discussion is beyond the scope of this brief review.

Neuroinflammation-induced lymphangiogenesis near the cribriform plate contributes to drainage of CNS-derived antigens and immune cells.

There are no conventional lymphatic vessels within the CNS parenchyma, although it has been hypothesized that lymphatics near the cribriform plate or dura maintain fluid homeostasis and immune surveillance during steady-state conditions. However, the role of these lymphatic vessels during neuroinflammation is not well understood. We report that lymphatic vessels near the cribriform plate undergo lymphangiogenesis in a VEGFC - VEGFR3 dependent manner during experimental autoimmune encephalomyelitis (EAE) and drain both CSF and cells that were once in the CNS parenchyma. Lymphangiogenesis also contributes to the drainage of CNS derived antigens that leads to antigen specific T cell proliferation in the draining lymph nodes during EAE. In contrast, meningeal lymphatics do not undergo lymphangiogenesis during EAE, suggesting heterogeneity in CNS lymphatics. We conclude that increased lymphangiogenesis near the cribriform plate can contribute to the management of neuroinflammation-induced fluid accumulation and immune surveillance.

Inflammatory macrophage migration in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a CD4 T cell-mediated demyelinating disease of the central nervous system (CNS) where macrophages are the end-stage effector cell. EAE serves as a model for multiple sclerosis where it has been instructive in delineating the autoimmune cellular response in the CNS for the purpose of developing more effective therapies. Understanding the nature of how cytokine and chemokine networks regulate the migration of leukocytes to the CNS requires the ability to track subpopulations of those cells in vivo. We describe a flow cytometric technique to monitor the migration of macrophages during EAE development.

Early embryonic lethality of mice with disrupted transcription cofactor PIMT/NCOA6IP/Tgs1 gene.

PIMT (also known as PIPMT/NCOA6IP/Tgs1), first isolated as a transcription coactivator PRIP (NCOA6)-interacting 96-kDa protein with RNA-binding property, possesses RNA methyltransferase activity. As a transcription coactivator binding protein, PIMT enhances the nuclear receptor transcriptional activity and its methyltransferase property is involved in the formation of the 2,2,7-trimethylguanosine cap of non-coding small RNAs, but the in vivo functions of this gene have not been fully explored. To elucidate the biological functions, we used gene targeting to generate mice with a disrupted PIMT/Tgs1 gene. Disruption of PIMT gene results in early embryonic lethality due to impairment of development around the blastocyst and uterine implantation stages. We show that PIMT is expressed in all cells of the E3.5day blastocyst in the mouse. PIMT null mutation abolished PIMT expression in all cells of the blastocyst and caused a reduction in the expression of Oct4 and Nanog transcription factor proteins in the E3.5 blastocyst resulting in the near failure to form inner cell mass (ICM). With conditional deletion of PIMT gene, mouse embryonic fibroblasts (MEFs) exhibit defective wound healing in the scratch assay and a reduction in cell proliferation due to decreased G0/G1 transition and G2/M phase cell cycle arrest. We conclude that PIMT/NCOA6IP, which is expressed in all cells of the 3.5 day stage blastocyst, is indispensable for early embryonic development.

Delta-like ligand 4 regulates central nervous system T cell accumulation during experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for multiple sclerosis. Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the current study, we hypothesized that Notch ligand-receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid dendritic cells, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) after EAE induction. Treatment of mice with a DLL4-specific blocking Ab significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by Ag-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of Ag-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished Ag-specific CD4(+) T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.

CCR4 contributes to the pathogenesis of experimental autoimmune encephalomyelitis by regulating inflammatory macrophage function.

Chemokines and their receptors play a critical role in orchestrating the immune response during experimental autoimmune encephalomyelitis (EAE). Expression of CCR4 and its ligand CCL22 has been observed in ongoing disease. Here we describe a role for CCR4 in EAE, illustrating delayed and decreased disease incidence in CCR4(-/-) mice corresponding with diminished CNS infiltrate. Peripheral T cell responses were unaltered in CCR4(-/-) mice; rather, disease reduction was related to reduced CD11b(+)Ly6C(hi) inflammatory macrophage (iMϕ) numbers and function. These results provide evidence that CCR4 regulates EAE development and further supports the involvement of CCR4 in iMϕ effector function.

CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function.

EAE is a demyelinating disease of the CNS and serves as a mouse model of MS. Expression of CCL22 in the draining LNs and spinal cord correlated with the onset of clinical EAE development and remained elevated. Administration of anti-CCL22 at the time of autoantigen immunization delayed the initiation of clinical disease and dampened the severity of peak initial disease and relapses. Reduced EAE severity correlated with the reduction of pathology and leukocytes in the CNS, particularly, activated CD11b+Ly6C(hi) macrophages. There were no differences in effector T cell-proliferative responses or effector T cell IFN-γ or IL-17 responses. However, treatment at the onset of disease did not reduce disease progression. Treatment of adoptive T cell transfer recipient mice with anti-CCL22 resulted in decreased clinical disease development accompanied by a decrease in CNS accumulation of CD11b+Ly6C(hi) macrophages. Neutralization of CCL22 resulted in a macrophage population whose effector cytokine expression consisted of decreased TNF and increased IL-10, a phenotype more consistent with M2 macrophages. This was corroborated by in vitro cultures of macrophages with CCL22. These results suggest that CCL22 functions to regulate development of EAE through macrophage chemoattraction and effector function.

IL-17-mediated monocyte migration occurs partially through CC chemokine ligand 2/monocyte chemoattractant protein-1 induction.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that is mediated, in part, by proinflammatory factors produced by RA synovial tissue (ST) fibroblasts and macrophages, resulting in monocyte migration from the blood to the ST. To characterize the potential role of IL-17 in monocyte migration, RA synovial fibroblasts and macrophages were activated with IL-17 and examined for the expression of monocyte chemokines. The two potentially important monocyte chemoattractants identified were CCL20/MIP-3alpha and CCL2/MCP-1, which were significantly induced in RA synovial fibroblasts and macrophages. However, in vivo, only CCL2/MCP-1 was detectable following adenovirus IL-17 injection. We found that IL-17 induction of CCL2/MCP-1 was mediated by the PI3K, ERK, and JNK pathways in RA ST fibroblasts and by the PI3K and ERK pathways in macrophages. Further, we show that neutralization of CCL2/MCP-1 significantly reduced IL-17-mediated monocyte recruitment into the peritoneal cavity. We demonstrate that local expression of IL-17 in ankle joints was associated with significantly increased monocyte migration and CCL2/MCP-1 levels. Interestingly, we show that RA synovial fluids immunoneutralized for IL-17 and CCL2/MCP-1 have similar monocyte chemotaxis activity as those immunoneutralized for each factor alone. In short, CCL2/MCP-1 produced from cell types present in the RA joint, as well as in experimental arthritis, may be responsible, in part, for IL-17-induced monocyte migration; hence, these results suggest that CCL2/MCP-1 is a downstream target of IL-17 that may be important in RA.

Experimental autoimmune encephalomyelitis in the mouse.

This unit details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Detailed materials and methods required for the purification of both PLP and MBP are also described. A protocol for isolating CNS-infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains.

Bing-Neel syndrome: an illustrative case and a comprehensive review of the published literature.

Waldenstrom's macroglobulinemia (WM) is a chronic lymphoproliferative disorder within the spectrum of lymphoplasmacytic lymphoma characterized by proliferation of plasma cells, small lymphocytes, and plasmacytoid lymphocytes. Central nervous system involvement is very rare (Bing-Neel [BN] syndrome). We present the case of a 62-year-old woman previously diagnosed with WM who presented with Bing-Neel syndrome and review the published literature which consists of only case reports. We performed a Medline search using the terms "Waldenstrom's macroglobulinemia and central nervous system" and "Bing-Neel" collecting data on presentation, evaluation, treatment, and outcome and summarizing these findings in the largest pooled series to date. Central nervous system manifestations are localization related. Serum laboratory testing reflects systemic disease. Cerebrospinal fluid analysis may show lymphocytic pleocytosis, elevated protein, and IgM kappa or lambda light chain restriction; cytology results are variable. Imaging is frequently abnormal. Biopsy confirms the diagnosis. Treatment data are limited, but responses are seen with radiation and/or chemotherapy. BN syndrome is a very rare complication of WM that should be considered in patients with neurologic symptoms and a history of WM. Treatment should be initiated as responses do occur that may improve quality of life and extend it when limited or no active systemic disease is present.

Beneficial role of the GPR30 agonist G-1 in an animal model of multiple sclerosis.

The beneficial effects of estrogens in multiple sclerosis are thought to be mediated exclusively by the classical nuclear estrogen receptors ERalpha and ERbeta. However, recently many reports revealed that estrogens are able to mediate rapid signals through a G protein-coupled receptor (GPCR), known as GPR30. In the present study, we set out to explore whether effects mediated through this receptor were anti-inflammatory and could account for some of the beneficial effects of estrogen. We demonstrate that GPR30 is expressed in both human and mouse immune cells. Furthermore a GPR30-selective agonist, G-1, previously described by us, inhibits the production of lipopolysaccharide (LPS)-induced cytokines such as TNF-alpha and IL-6 in a dose-dependent manner in human primary macrophages and in a murine macrophage cell line. These effects are likely mediated solely through the estrogen-specific receptor GPR30 since the agonist G-1 displayed an IC(50) far greater than 10 microM on the classical nuclear estrogen receptors as well as a panel of 25 other GPCRs. Finally, we show that the agonist G-1 is able to reduce the severity of disease in both active and passive EAE models of multiple sclerosis in SJL mice and that this effect is concomitant with a G-1-mediated decrease in proinflammatory cytokines, including IFN-gamma and IL-17, in immune cells harvested from these mice. The effect of G-1 appears indirect, as the GPR30 agonist did not directly influence IFN-gamma or IL-17 production by purified T cells. These data indicate that G-1 may represent a novel therapeutic agent for the treatment of chronic autoimmune, inflammatory diseases.

Mice deficient for CCR6 fail to control chronic experimental autoimmune encephalomyelitis.

Chemokines are a superfamily of chemotactic cytokines that play an important role in leukocyte trafficking and have been implicated as functional mediators of immunopathology in experimental autoimmune encephalomyelitis (EAE). In the present study, we investigated the role of the CCL20 receptor, CCR6, in chronic EAE. After immunization with myelin oligodendrocyte glycoprotein 35-55 in CFA, CCR6(-/-) mice developed a significantly more severe chronic EAE as compared to wild type immunized animals. CCR6 expression was not required by T cells to induce EAE. Measurement of peripheral T cell responses showed differences in IFN-gamma and IL-17 responses between CCR6(-/-) and wild type mice. At the time when CCR6(-/-) mice showed significantly more severe chronic EAE there was a significant decrease in PD-L1-expressing mDC in the spleens and no differences in Foxp3 Treg. Furthermore, add back of mDC with increased PD-L1 expression to CCR6(-/-) mice reduced the severe chronic EAE disease phase to that of wild type controls. The results suggest a role for CCR6-expressing PDL1(+) mDC in regulating EAE progression.

MCP-1-deficient mice show reduced neuroinflammatory responses and increased peripheral inflammatory responses to peripheral endotoxin insult.

BACKGROUND: An endotoxin insult mimics a severe peripheral infection and recent evidence suggests that a single exposure can cause long-term cognitive deficits. A peripheral injection of LPS results in production of pro-inflammatory cytokines, such as IL-1beta and TNF-alpha, in the brain and periphery and these cytokines mediate many effects of the acute phase response including activation of the HPA axis. The chemokine MCP-1 is highly expressed during endotoxemia and although much is known about the importance of MCP-1 in peripheral inflammatory responses to LPS, information about MCP-1 and CNS responses to peripheral LPS is lacking. METHODS: C57Bl/6 mice were administered LPS by intraperitoneal (i.p.) injection, serum and brains were collected at several time points, and the time course of MCP-1 protein up-regulation was measured. To examine the role of MCP-1 in activation of the brain during acute systemic inflammation, we injected MCP-1 knockout (MCP-1-/-) or control C57Bl/6 (MCP-1+/+) mice with LPS i.p. and measured the levels of selected cytokines and chemokines in serum and brain extracts 6 hours later. Activated microglia were examined by CD45 immunohistochemistry, and serum corticosterone and ACTH levels were measured by enzyme immunoassay. RESULTS: We report that LPS injection induces a robust increase in MCP-1 protein levels in serum and brain, with peak brain levels reached at 6 hrs after LPS administration. MCP-1-/- mice injected with LPS showed higher levels of serum IL-1beta and TNF-alpha compared to LPS-treated MCP-1+/+ mice. In contrast, these MCP-1-/- mice showed significantly lower inductions of brain pro-inflammatory cytokines and chemokines, fewer activated microglia, and a reduction in serum corticosterone levels. CONCLUSION: MCP-1-/- mice have decreased brain inflammation after a peripheral LPS insult, despite an exaggerated peripheral response. These data demonstrate an important role for MCP-1 in regulation of brain inflammation after peripheral endotoxemia.

Inhibition of experimental autoimmune encephalomyelitis by a novel small molecular weight proinflammatory cytokine suppressing drug.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated, autoimmune disease of the central nervous system (CNS) that serves as a model for various cellular and molecular aspects of the human disease, multiple sclerosis (MS). Although EAE has long been considered a T cell-mediated disease, macrophages play a role in disease pathogenesis and are known to accumulate in the CNS under the control of chemokines. In the present report we demonstrate that mice induced to develop EAE were treated with a small molecular weight molecule that suppresses proinflammatory cytokine production which resulted in significantly decreased clinical EAE, CNS CCL2 expression and CNS macrophage accumulation. These results demonstrate the efficacy of a novel class of therapeutic molecules for CNS demyelinating disease.

Production of CCL2 by central nervous system cells regulates development of murine experimental autoimmune encephalomyelitis through the recruitment of TNF- and iNOS-expressing macrophages and myeloid dendritic cells.

Experimental autoimmune encephalomyelitis is a T cell-mediated demyelinating disease of the CNS that serves as a model for the human disease multiple sclerosis. Increased expression of the chemokine CCL2 in the CNS has been demonstrated to be important in the development of demyelinating disease presumably by attracting inflammatory cells. However, the mechanism of how CCL2 regulates disease pathogenesis has not been fully elucidated. Using radiation bone marrow chimeric mice we demonstrated that optimum disease was achieved when CCL2 was glia derived. Furthermore, CNS production of CCL2 resulted in the accumulation of iNOS-producing CD11b(+)CD11c(+) dendritic cells and TNF-producing macrophages important for demyelination. Lack of glial-derived CCL2 production did not influence experimental autoimmune encephalomyelitis by altering either Th1 or Th17 cells, as there were no differences in these populations in the CNS or periphery between groups. These results demonstrate that the glial-derived CCL2 is important for the attraction of TNF- and iNOS-producing dendritic cells and effector macrophages to the CNS for development of subsequent autoimmune disease.

CCR2 regulates development of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a murine model for multiple sclerosis, involves recruitment of T cells and macrophages to the CNS after infection. We hypothesized that CCR2, the only known receptor for CCL2, would be required for TMEV-induced demyelinating disease development because of its role in macrophage recruitment. TMEV-infected SJL CCR2 knockout (KO) mice showed decreased long-term clinical disease severity and less demyelination compared with controls. Flow cytometric data indicated that macrophages (CD45(high) CD11b(+) ) in the CNS of TMEV-infected CCR2 KO mice were decreased compared with control mice throughout disease. CD4(+) and CD8(+) T cell percentages in the CNS of TMEV-infected control and CCR2 KO mice were similar over the course of disease. There were no apparent differences between CCR2 KO and control peripheral immune responses. The frequency of interferon-gamma-producing T cells in response to proteolipid protein 139-151 in the CNS was also similar during the autoimmunity stage of TMEV-induced demyelinating disease. These data suggest that CCR2 is important for development of clinical disease by regulating macrophage accumulation after TMEV infection.

Critical role for transcription coactivator peroxisome proliferator-activated receptor (PPAR)-binding protein/TRAP220 in liver regeneration and PPARalpha ligand-induced liver tumor development.

Disruption of the gene encoding for the transcription coactivator peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205/Med1) in the mouse results in embryonic lethality. Here, we have reported that targeted disruption of the Pbp/Pparbp gene in hepatocytes (Pbp(DeltaLiv)) impairs liver regeneration with low survival after partial hepatectomy. Analysis of cell cycle progression suggests a defective exit from quiescence, reduced BrdUrd incorporation, and diminished entry into G(2)/M phase in Pbp(DeltaLiv) hepatocytes after partial hepatectomy. Pbp(DeltaLiv) hepatocytes failed to respond to hepatocyte growth factor/scatter factor, implying that hepatic PBP deficiency affects c-met signaling. Pbp gene disruption also abolishes primary mitogen-induced liver cell proliferative response. Striking abrogation of CCl(4)-induced hepatocellular proliferation and hepatotoxicity occurred in Pbp(DeltaLiv) mice pretreated with phenobarbital due to lack of expression of xenobiotic metabolizing enzymes necessary for CCl(4) activation. Pbp(DeltaLiv) mice, chronically exposed to Wy-14,643, a PPARalpha ligand, revealed a striking proliferative response and clonal expansion of a few Pbp(fl/fl) hepatocytes that escaped Cre-mediated gene deletion in Pbp(DeltaLiv) livers, but no proliferative expansion of PBP null hepatocytes was observed. In these Pbp(DeltaLiv) mice, none of the Wy-14,643-induced hepatic adenomas and hepatocellular carcinomas was derived from PBP(DeltaLiv) hepatocytes; all liver tumors developing in Pbp(DeltaLiv) mice maintained non-recombinant Pbp alleles and retained PBP expression. These studies provide direct evidence in support of a critical role of PBP/TRAP220 in liver regeneration, induction of hepatotoxicity, and hepatocarcinogenesis.