Ninjurin1 Deficiency Attenuates Susceptibility of Experimental Autoimmune Encephalomyelitis in Mice

Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, in vivo gene deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knock-out (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis. Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in endotoxin-induced uveitis mice. Moreover, the administration of a custom-made antibody (Ab_26–37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone marrow-derived macrophages and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO bone marrow-derived macrophages and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1-overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as multiple sclerosis.     

Analysis of the Gadolinium retention in the Experimental Autoimmune Encephalomyelitis (EAE) murine model of Multiple Sclerosis

ObjectivesThe aim of this study is to quantitatively investigate, at the preclinical level, the extent of Gd retention in the CNS, and peripheral organs, of immune-mediated murine models (Experimental Autoimmune Encephalomyelitis –EAE) of Multiple Sclerosis, compared to control animals, upon the injection of gadodiamide. The influence of the Gadolinium Based Contrast Agent administration timing during the course of EAE development is also monitored.MethodsEAE mice were injected with three doses (1.2 mmol/kg each) of gadodiamide at three different time points during the EAE development and sacrificed after 21 or 39 days. Organs were collected and the amount of Gd was quantified through Inductively Coupled Plasma-Mass Spectrometry. Transmission electron microscopy (TEM) and MRI techniques were applied to add spatial and qualitative information to the obtained results.ResultsIn the spinal cord of EAE group, 21 days after gadodiamide administration, a significantly higher accumulation of Gd occurred. Conversely, in the encephalon, a lower amount of Gd retention was reached, even if differences emerged between EAE and controls mice. After 39 days, the amounts of retained Gd markedly decreased. TEM validated the presence of Gd in CNS. MRI of the encephalon at 7.1T did not highlight any hyper intense region.ConclusionIn the spinal cord of EAE mice, which is the mostly damaged region in this specific animal model, a preferential but transient accumulation of Gd is observed. In the encephalon, the Gd retention could be mostly related to inflammation occurring upon immunization rather than to demyelination.     

C57BL/6J小鼠实验性自身免疫性脑脊髓炎模型的建立及其病理特征

目的探讨C57BL/6J小鼠建立实验性自身免疫性脑脊髓炎（EAE）模型的可能性及其发病特点。方法使用PLP139-151抗原及其C57BL/6J小鼠自制脑脊髓匀浆（spinal cord homogenate,SCH）免疫C57BL/6J小鼠,使用完全福（氏）免疫佐剂为免疫佐剂,并在尾静脉注射百日咳杆菌,建立EAE模型,与经典的PLP139-151免疫的SJL/J小鼠EAE模型进行对比。结果PLP139-151免疫C57BL/6J小鼠仅有一只小鼠表现为尾部张力明显降低;自制SCH免疫C57BL/6J小鼠可见明显脱髓鞘改变。与PLP139-151免疫SJL/J小鼠组相比发病率较低（P〈0.05）,神经功能评分比较没有明显差异（P〉0.05）,但发病时间长于PLP139-151免疫SJL/J小鼠组（P〈0.05）。结论SCH免疫C57BL/6J小鼠的EAE动物模型,主要表现为急性单相病程,从临床表现和病理学特点来看符合人类MS的病理特点,值得在以后的研究中进一步研究探讨。     

Interleukin-17 (IL-17)-induced MicroRNA 873 (miR-873) Contributes to the Pathogenesis of Experimental Autoimmune Encephalomyelitis by Targeting A20 Ubiquitin-editing Enzyme

Interleukin 17 (IL-17), produced mainly by T helper 17 (Th17) cells, is increasingly recognized as a key regulator in various autoimmune diseases, including human multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although several microRNAs (miRNAs) with aberrant expression have been shown to contribute to the pathogenesis of MS and EAE, the mechanisms underlying the regulation of abnormal miRNA expression in astrocytes upon IL-17 stimulation remain unclear. In the present study, we detected the changes of miRNA expression profiles both in the brain tissue of EAE mice and in cultured mouse primary astrocytes stimulated with IL-17 and identified miR-873 as one of the co-up-regulated miRNAs in vivo and in vitro. The overexpression of miR-873, demonstrated by targeting A20 (TNFα-induced protein 3, TNFAIP3), remarkably reduced the A20 level and promoted NF-κB activation in vivo and in vitro as well as increasing the production of inflammatory cytokines and chemokines (i.e. IL-6, TNF-α, MIP-2, and MCP-1/5). More importantly, silencing the endogenous miR-873 or A20 gene with lentiviral vector of miR-873 sponge (LV-miR-873 sponge) or short hairpin RNA (shRNA) of A20 (LV-A20 shRNA) in vivo significantly lessened or aggravated inflammation and demyelination in the central nervous system (CNS) of EAE mice, respectively. Taken together, these findings indicate that miR-873 induced by IL-17 stimulation promotes the production of inflammatory cytokines and aggravates the pathological process of EAE mice through the A20/NF-κB pathway, which provides a new insight into the mechanism of inflammatory damage in MS.     

AKP-11 - A Novel S1P1 Agonist with Favorable Safety Profile Attenuates Experimental Autoimmune Encephalomyelitis in Rat Model of Multiple Sclerosis

Sphingosine-1-phosphate receptor 1 (S1P1) mediated regulation of lymphocyte egress from lymphoid organs is recognized as the mechanism of FTY720 (Fingolimod, Gilenya) efficacy in relapsing-remitting forms of multiple sclerosis (RRMS). In this study we describe a novel S1P1 agonist AKP-11, next generation of S1P1 agonist, with immunomodulatory activities in cell culture model and for therapeutic efficacy against an animal model of MS, i.e. experimental autoimmune encephalomyelitis (EAE) but without the adverse effects observed with FTY720. Like FTY720, AKP-11 bound to S1P1 is internalized and activates intracellular AKT and ERKs cellular signaling pathways. In contrast to FTY720, AKP-11 mediated S1P1 downregulation is independent of sphingosine kinase activity indicating it to be a direct agonist of S1P1. The S1P1 loss and inhibition of lymphocyte egress by FTY720 leads to lymphopenia. In comparison with FTY720, oral administration of AKP-11 caused milder and reversible lymphopenia while providing a similar degree of therapeutic efficacy in the EAE animal model. Consistent with the observed reversible lymphopenia with AKP-11, the S1P1 recycled back to cell membrane in AKP-11 treated cells following its withdrawal, but not with withdrawal of FTY720. Accordingly, a smaller degree of ubiquitination and proteolysis of S1P1 was observed in AKP-11 treated cells as compared to FTY720. Consistent with previous observations, FTY720 treatment is associated with adverse effects of bradycardia and lung vascular leaks in rodents, whereas AKP-11 treatment had undetectable effects on bradycardia and reduced lung vascular leaks as compared to FTY720. Taken together, the data documents that AKP-11 treatment cause milder and reversible lymphopenia with milder adverse effects while maintaining therapeutic efficacy similar to that observed with FTY720, thus indicating therapeutic potential of AKP-11 for treatment of MS and related autoimmune disorders.     

Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya alata

Coumarin and its derivatives are fragrant natural compounds isolated from the genus Murraya that are flowering plants widely distributed in East Asia, Australia, and the Pacific Islands. Murraya plants have been widely used as medicinal herbs for relief of pain, such as headache, rheumatic pain, toothache, and snake bites. However, little is known about their analgesic components and the molecular mechanism underlying pain relief. Here, we report the bioassay-guided fractionation and identification of a novel coumarin derivative, named muralatin L, that can specifically activate the nociceptor transient receptor potential vanilloid 1 (TRPV1) channel and reverse the inflammatory pain in mice through channel desensitization. Muralatin L was identified from the active extract of Murraya alata against TRPV1 transiently expressed in HEK-293T cells in fluorescent calcium FlexStation assay. Activation of TRPV1 current by muralatin L and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV1-expressing HEK-293T cells and dorsal root ganglion neurons isolated from mice. Furthermore, muralatin L could reverse inflammatory pain induced by formalin and acetic acid in mice but not in TRPV1 knock-out mice. Taken together, our findings show that muralatin L specifically activates TRPV1 and reverses inflammatory pain, thus highlighting the potential of coumarin derivatives from Murraya plants for pharmaceutical and medicinal applications such as pain therapy.