Berberine attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice

Background

Berberine, an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-inflammatory and neuroprotective effects. However, there are no reports about the effects and mechanisms of berberine in experimental autoimmune encephalomyelitis (EAE), an established model of multiple sclerosis (MS).

Methodology/Principal Findings

Female C57 BL/6 mice immunized with myelin oligodendrocyte glycoprotein 35–55 amino acid peptide were treated with berberine at the day of disease onset and medication was administered daily until mice were sacrificed. Blood–brain barrier (BBB) permeability and the alteration of matrix metalloproteinase-2 (MMP-2, 72 kDa) and matrix metalloproteinase-9 (MMP-9, 92 kDa) in the brain and cerebrospinal fluid (CSF) of EAE mice were detected by quantitative measurement for Evan''s blue (EB) content, Western blot and gelatin zymography respectively. The results showed that berberine attenuated clinical and pathological parameters of EAE, reduced the permeability of BBB, inhibited the activity and expression of MMP-9 but not MMP-2 in the CSF and brain of EAE mice.

Conclusions/Significance

These findings suggest that berberine is effective to attenuate the clinical severity of EAE in C57 BL/6 mice by reducing the permeability of BBB, decreasing the expression and activity of MMP-9, and decreasing the inflammatory infiltration. We think that berberine might be a potential therapeutic agent for MS.     

SAP suppresses the development of experimental autoimmune encephalomyelitis in C57BL/6 mice

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated disease of the central nervous system. Serum amyloid P component (SAP) is a highly conserved plasma protein named for its universal presence in amyloid deposits. Here we report that SAP-transgenic mice had unexpectedly attenuated EAE due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35-55 in complete Freund's adjuvant, SAP-transgenic mice showed reduced spinal cord inflammation with lower severity of EAE attacks as compared with control C57BL/6 mice. However, in SAP-Knockout mice, the severity of EAE is enhanced. Adoptive transfer of Ag-restimulated T cells from wild type to SAP-transgenic mice, or transfer of SAP-transgenic Ag-restimulated T cells to control mice, induced milder EAE. T cells from MOG-primed SAP-transgenic mice showed weak proliferative responses. Furthermore, in SAP-transgenic mice, there is little infiltration of CD45-positive cells in the spinal cord. In vitro, SAP suppressed the secretion of interleukin-2 stimulated by P-selectin and blocked P-selectin binding to T cells. Moreover, SAP could change the affinity between α4-integrin and T cells. These data suggested that SAP could antagonize the development of the acute phase of inflammation accompanying EAE by modulating the function of P-selectin.     

Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by photobiomodulation induced by 670 nm light

Background

The approved immunomodulatory agents for the treatment of multiple sclerosis (MS) are only partially effective. It is thought that the combination of immunomodulatory and neuroprotective strategies is necessary to prevent or reverse disease progression. Irradiation with far red/near infrared light, termed photobiomodulation, is a therapeutic approach for inflammatory and neurodegenerative diseases. Data suggests that near-infrared light functions through neuroprotective and anti-inflammatory mechanisms. We sought to investigate the clinical effect of photobiomodulation in the Experimental Autoimmune Encephalomyelitis (EAE) model of multiple sclerosis.

Methodology/Principal Findings

The clinical effect of photobiomodulation induced by 670 nm light was investigated in the C57BL/6 mouse model of EAE. Disease was induced with myelin oligodendrocyte glycoprotein (MOG) according to standard laboratory protocol. Mice received 670 nm light or no light treatment (sham) administered as suppression and treatment protocols. 670 nm light reduced disease severity with both protocols compared to sham treated mice. Disease amelioration was associated with down-regulation of proinflammatory cytokines (interferon-γ, tumor necrosis factor-α) and up-regulation of anti-inflammatory cytokines (IL-4, IL-10) in vitro and in vivo.

Conclusion/Significance

These studies document the therapeutic potential of photobiomodulation with 670 nm light in the EAE model, in part through modulation of the immune response.     

Gamma delta T cell regulation of IFN-gamma production by central nervous system-infiltrating encephalitogenic T cells: correlation with recovery from experimental autoimmune encephalomyelitis

Interferon-gamma has been shown to be important for the resolution of inflammation associated with CNS autoimmunity. Because one of the roles of gamma delta T cells is the regulation of inflammation, we asked whether gamma delta T cells were able to regulate CNS inflammation using the autoimmune disease mouse model experimental autoimmune encephalomyelitis (EAE). We show that the presence of gamma delta T cells was needed to promote the production of IFN-gamma by both CD4 and CD8 T cells in the CNS before the onset of EAE. This regulation was shown to be independent of the ability of gamma delta T cells to produce IFN-gamma, and was specific to T cells in the CNS, as no alterations in IFN-gamma production were detectable in gamma delta T cell-deficient mice in the spleen and lymph nodes of mice with EAE or following immunization. Analysis of TCR gamma delta gene usage in the CNS showed that the only TCR delta V gene families present in the CNS before EAE onset are from the DV7s6 and DV105s1 gene families. We also show that the primary IFN-gamma-producing cells in the CNS are the encephalitogenic T cells, and that gamma delta T cell-deficient mice are unable to resolve EAE disease symptoms like control mice, thus exhibiting a long-term chronic disease course similar to that observed in IFN-gamma-deficient mice. These data suggest that CNS resident gamma delta T cells promote the production of IFN-gamma by encephalitogenic T cells in the CNS, which is ultimately required for the recovery from EAE.     

Rat and human myelin oligodendrocyte glycoproteins induce experimental autoimmune encephalomyelitis by different mechanisms in C57BL/6 mice

C57BL/6 mice immunized with the extracellular Ig-like domain of rat myelin oligodendrocyte glycoprotein (MOG) developed experimental autoimmune encephalomyelitis (EAE) resembling that induced by rodent MOG 35-55 in its B cell independence and predominantly mononuclear CNS infiltrate. In contrast, human MOG protein-induced EAE was B cell dependent with polymorphonuclear leukocytes. Human MOG differs from rat MOG at several residues, including a proline for serine substitution at position 42. Human MOG 35-55 was only weakly encephalitogenic, and a proline substitution in rat MOG at position 42 severely attenuated its encephalitogenicity. However, human MOG 35-55 was immunogenic, inducing proliferation and IFN-gamma and IL-13 to human, but not rodent MOG 35-55 [corrected]. The B cell dependence of EAE induced by human MOG protein was not due to a requirement for Ag presentation by B cells, because spleen cells from B cell-deficient mice processed and presented human and rat MOG proteins to T cells. The different pathogenic mechanisms of human and rat MOG proteins might result from different Abs induced by these proteins. However, rat and human MOG proteins induced Abs to mouse MOG that were equivalent in titer and IgG subclass. These data demonstrate that EAE can be induced in C57BL/6 mice by two mechanisms, depending on the nature of the immunogen: an encephalitogenic T cell response to rat MOG or rodent MOG 35-55, or an encephalitogenic B cell response to epitopes on human MOG protein that most likely cross-react with mouse determinants.     

Extensive infiltration of neutrophils in the acute phase of experimental autoimmune encephalomyelitis in C57BL/6 mice

To determine the possible involvement of neutrophils in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), we examined their infiltration pattern during the course of MOG_35–55-induced EAE in the C57BL/6 mice. Using immunohistochemistry and flow cytometry, we found that the number of neutrophils was significantly increased during onset of disease, remained high at the peak stage and dramatically declined thereafter. Moreover, dual labeling provided anatomical evidence of a prominent accumulation of neutrophils in the center and vicinity of lesion areas of demyelination, axonal loss or axonal degeneration at early stages of EAE. These observations provide evidence that neutrophils are one of the major sources of inflammatory cells to initiate EAE, which suggest that neutrophils may contribute to demyelination and axonal degeneration in the acute phase of EAE and play a greater role than previously thought in the pathogenesis of EAE.     

Influenza vaccine and learning in C57BL mice with an acute experimental autoimmune encephalomyelitis

Literature data suggest possible link between influenza vaccination and development of autoimmune processes. Therefore, the aim of the study was to investigate the effect of influenza vaccination on spatial learning in mice with experimental autoimmune encephalomyelitis (EAE). EAE was induced in eight-week-old C57BL/6J female mice by subcutaneous immunization (MOG_35–55 in complete Freund’s adjuvant) and Pertussis vaccine injected intraperitoneally. Mice were vaccinated with influenza vaccine three days before MOG immunization. The hippocampal-dependent spatial learning test, Morris Water Maze test (MWM), was performed before and after EAE induction. Significant difference (P < 0.05) in the time for completing the Morris Water Maze task was found between mice with mild clinical signs of EAE when compared to other mice. However no significant difference was observed between mice with EAE and mice with EAE that were vaccinated with influenza vaccine. Hippocampal tissue lesions in EAE mice are in correlation with memory impairment. Study shows no influence of influenza vaccine on progression of clinical signs of EAE, spatial learning and memory impairment.     

E- and P-selectin are not required for the development of experimental autoimmune encephalomyelitis in C57BL/6 and SJL mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier (BBB) and gain access to the CNS. It is well-established that alpha4 integrins are actively involved in leukocyte recruitment across the BBB during EAE. In contrast, the role of endothelial E- and P-selectin in this process has been a controversial issue. In this study, we demonstrate that P-selectin protein can be detected in meningeal blood vessel endothelial cells in healthy SJL and C57BL/6 mice and on rare parenchymal CNS blood vessels in C57BL/6, but not SJL, mice. During EAE, expression of P-selectin but not E-selectin was found up-regulated on inflamed CNS microvessels surrounded by inflammatory infiltrates irrespective of their meningeal or parenchymal localization with a more prominent immunostaining detected in C57BL/6 as compared with SJL mice. P-selectin immunostaining could be localized to CNS endothelial cells and to CD41-positive platelets adhering to the vessel wall. Despite the presence of P-selectin in wild-type mice, E/P-selectin-deficient SJL and C57BL/6 mice developed clinical EAE indistinguishable from wild-type mice. Absence of E- and P-selectin did neither influence the activation of myelin-specific T cells nor the composition of the cellular infiltrates in the CNS during EAE. Finally, endothelial-specific tetracycline-inducible expression of E-selectin at the BBB in transgenic C57BL/6 mice did not alter the development of EAE. Thus, E- and P-selectin are not required for leukocyte recruitment across the BBB and the development of EAE in C57BL/6 and in SJL mice.     

P-selectin glycoprotein ligand 1 is not required for the development of experimental autoimmune encephalomyelitis in SJL and C57BL/6 mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier and gain access to the CNS. The involvement of P-selectin glycoprotein ligand 1 (PSGL-1) and of its major endothelial ligand P-selectin in this process have been controversial. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1, which can bind to soluble and immobilize P-selectin if presented in high concentrations, PSGL-1 is not involved T cell interaction with P-selectin expressing brain endothelial cells in vitro. Furthermore, neither anti-PSGL-1 Abs nor the lack of PSGL-1 in PSGL-1-deficient mice inhibits the recruitment of inflammatory cells across the blood-brain barrier or the development of clinical EAE. Taken together, our findings demonstrate that PSGL-1 is not required for the pathogenesis of EAE.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Intrathecal Fas ligand infusion strengthens immunoprivilege of central nervous system and suppresses experimental autoimmune encephalomyelitis

Fas ligand (FasL) is an essential molecule strongly expressed in some immunoprivileged sites, but is expressed at very low levels in normal CNS. In this study, acute experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats with guinea pig myelin basic protein. Intrathecal infusion of recombinant FasL before EAE onset dose dependently suppressed acute EAE and alleviated pathological inflammation in lumbosacral spinal cord. This treatment greatly increased apoptosis in CNS inflammatory cells, but did not inhibit systemic immune response to myelin basic protein. Systemic administration of a similar dose of rFasL was ineffective. In vitro, encephalitogenic T cells were highly sensitive to rFasL-induced cell death, and activated macrophages were also susceptible. In addition, in vitro rFasL treatment potentiated the immunosuppressive property of rat cerebrospinal fluid. We conclude that intrathecal infusion of rFasL eliminated the initial wave of infiltrating T cells and macrophages, and therefore blocked the later recruitment of inflammatory cells into CNS. Although Fas receptor expression was observed on spinal cord neurons, astrocytes, and oligodendrocytes, no damage to these cells or to the myelin structure was detected after rFasL infusion.     

Absence of IFN-gamma or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.     

Collagenase-3 (MMP-13) deficiency protects C57BL/6 mice from antibody-induced arthritis

Introduction

Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis.

Methods

For this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13^–/–) mice by intraperitoneal injection of 200 μl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice.

Results

This study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13^–/– mice developed progressive arthritis with a similar onset. However, MMP-13^–/– mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13^–/– mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods.

Conclusions

MMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target.     

Conjugated linoleic acid protects against age-associated bone loss in C57BL/6 female mice

Osteoporosis is one of the major causes of morbidity in the elderly. Inflammation exerts a significant influence on bone turnover, inducing the chronic form of osteoporosis. Dietary nutrition has the capacity to modulate inflammatory response. Therefore, nutritional strategies and lifestyle changes may prevent age-related osteoporosis, thereby improving the quality of life of the elderly population. Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Hence, this study was undertaken to examine the effect of CLA on bone mineral density (BMD) in middle-aged C57BL/6 female mice. After 10 weeks on diet, CLA-fed mice (14 months) maintained a higher BMD in different bone regions than corn oil (CO)-fed mice. The increased BMD was accompanied by a decreased activity of proinflammatory cytokines (such as tumor necrosis factor alpha, interleukin-6 and the receptor activator of NF-kappaB ligand) and decreased osteoclast function. Furthermore, a significant decrease in fat mass and an increase in muscle mass were also observed in CLA-fed mice compared to CO-fed mice. In conclusion, these findings suggest that CLA may prevent the loss of bone and muscle mass by modulating markers of inflammation and osteoclastogenic factors.     

Differences in anxiety-related behavior between apolipoprotein E-deficient C57BL/6 and wild type C57BL/6 mice

The influence of ApoE gene deletion on the anxiety state has not been previously investigated. The elevated plus maze was used in this study to determine differences in anxiety-related behavior between apoE-deficient and wild type C57BL/6 mice. The apoE-deficient mice demonstrated less anxiety on the elevated plus maze by spending more time in the open arms of the elevated plus maze compared to wild type mice (p<0.001). Additionally, female apoE-deficient mice visited the open arm of the maze more often than their apoE-deficient male counterpart (p<0.05). The anxiety state and/or sex are possible variables to be considered when designing physiological and/or behavioral studies involving mice that are apoE-deficient.     

Fertilizing ability of spermatozoa from aged C57BL/6NNia mice

Spermatozoa from C57BL/6NNia mice (7- and 25-month-old males that produced offspring and 25-month-old males incapable of producing offspring which either mated or did not mate after being paired for 1 month with proven-fertile females) were tested in in-vitro fertilization studies. The 7-month-old males fertilized the largest number of oocytes (80-86%) in vitro and 79% of them subsequently developed into blastocysts in culture. Aged males which failed to mate fertilized the lowest number of oocytes (11-19%) with 48% developing to blastocysts. This group of mice had the lowest number of spermatozoa in the cauda epididymidis (3.2 +/- 0.4 x 10(5)/mg tissue) with fewer motile spermatozoa (22.3 +/- 5.1%) than younger males. The percentage of spermatozoa retaining their acrosome after 3 h in culture was higher in aged males which had not mated when compared to younger males that had mated. After 4 h in culture, however, the number of spermatozoa that had lost their acrosome was almost identical in the two groups. Superovulated mice which were artificially inseminated with spermatozoa from 25-month-old mice that had not mated did not become pregnant. Testosterone concentrations were lowest in aged mice not mating. These concentrations may explain the poor behavioural response of these males, but whether they account for the inability of spermatozoa to fertilize ova in vitro or in vivo after artificial insemination is not known.     

Regulation of experimental autoimmune encephalomyelitis in the C57BL/6J mouse by NK1.1+, DX5+, alpha beta+ T cells

C57BL/6 (B6) mice with targeted mutations of immune function genes were used to investigate the mechanism of recovery from experimental autoimmune encephalomyelitis (EAE). The acute phase of passive EAE in the B6 mouse is normally resolved by partial recovery followed by mild sporadic relapses. B6 TCR beta-chain knockout (KO) recipients of a myelin oligodendrocyte glycoprotein p35-55 encephalitogenic T cell line failed to recover from the acute phase of passive EAE. In comparison with wild-type mice, active disease was more severe in beta(2)-microglobulin KO mice. Reconstitution of TCR beta-chain KO mice with wild-type spleen cells halted progression of disease and favored recovery. Spleen cells from T cell-deficient mice, IL-7R KO mice, or IFN-gamma KO mice were ineffective in this regard. Irradiation or treatment of wild-type spleen cell population with anti-NK1.1 mAb before transfer abrogated the protective effect. Removal of DX5(+) cells from wild-type spleen cells by anti-DX5 Ab-coated magnetic beads before reconstitution abrogated the suppressive properties of the spleen cells. TCR-deficient recipients of the enriched DX5(+) cell population recovered normally from passively induced acute disease. DX5(+) cells were sorted by FACS into DX5(+) alpha beta TCR(+) and DX5(+) alpha beta TCR(-) populations. Only recipients of the former recovered normally from clinical disease. These results indicate that recovery from acute EAE is an active process that requires NK1.1(+), DX5(+) alpha beta(+) TCR spleen cells and IFN-gamma.     

Splenic eumelanin differs from hair eumelanin in C57BL/6 mice

The presence of melanin in spleens of black C57BL/6 mice has been known for long. Although its origin and biological functions are still obscure, the relation of splenic melanin to the hair follicle and skin pigmentation was suggested. Here, we demonstrated using for the first time electron paramagnetic resonance spectroscopy that black-spotted C57BL/6 spleens contain eumelanin. Its presence here is a "yes or no" phenomenon, as even in the groups which revealed the highest percentage of spots single organs completely devoid of the pigment were found. Percentage of the spotted spleens decreased, however, with the progress of telogen after spontaneously-induced hair growth. The paramagnetic properties of the spleen eumelanin differed from the hair shaft or anagen VI skin melanin. The splenic melanin revealed narrower signal, and its microwave power saturability betrayed more heterogenous population of paramagnetic centres than in the skin or hair shaft pigment. Interestingly, the pigment of dry hair shafts and of the wet tissue of depilated anagen VI skin revealed almost identical properties. The properties of splenic melanin better resembled the synthetic dopa melanin (water suspension, and to a lesser degree - powder sample) than the skin/hair melanin. All these findings may indicate a limited degradation of splenic melanin as compared to the skin/hair pigment. The splenic eumelanin may at least in part originate from the skin melanin phagocyted in catagen by the Langerhans cells or macrophages and transported to the organ.