Experimental Autoimmune Encephalitis Model Research Articles

Carbenoxolone mitigates extensive fibrosis formation in PLP-induced EAE model and multiple sclerosis serum-exposed pericyte culture.

Multiple sclerosis (MS) is one of the most common causes of disability in young adults. Nearly, 85% of MS cases start with attacks and remissions, classified as relapsing-remitting multiple sclerosis (RRMS). With repeating attacks, MS causes brain-spinal cord atrophy and enhanced disability as disease progresses. PLP-induced EAE is one of the most established models for pathophysiology and treatment of RRMS. Recent studies demonstrated the possible role of pericytes in perivascular and intra-lesional fibrosis in PLP-induced EAE, whose importance remains elusive. Hence, we have investigated the possible role of pericytes in fibrosis formation and amelioration with a hemichannel blocker, Carbenoxolone (CBX). PLP-induced experimental autoimmune encephalitis (EAE) model is used and the effect of CBX is investigated. Clinical scores were recorded and followed. Perivascular Collagen 1 and 3 accumulations were demonstrated as markers of fibrosis in the spinal cord. To delineate the role of pericytes, human brain vascular pericytes (HBVP) were incubated with the sera of MS patients to induce in-vitro MS model and the fibrosis formation was investigated. In the PLP induced in-vivo model, both intracerebroventricular and intraperitoneal CBX have significantly mitigated the disease progression followed by clinical scores, demyelination, and fibrosis. Moreover, CBX significantly mitigated MS-serum-induced fibrosis in the HBVP cell culture. The study demonstrated two important findings. First, CBX decreases fibrosis formation in both in-vivo and in-vitro MS models. Secondly, it improves neurological scores and decreases demyelination in the EAE model. Therefore, CBX can be potential novel therapeutic option in treating Multiple Sclerosis.

Dabrafenib mitigates the neuroinflammation caused by ferroptosis in experimental autoimmune encephalomyelitis by up regulating Axl receptor

Multiple sclerosis is an autoimmune disease that causes inflammatory damage to the central nervous system. At present, the pathogenesis of the disease is unknown. There is a lack of few effective therapy medications available. Therefore, it is necessary to further explore the pathogenesis of this illness and develop potential therapeutic drugs. Dabrafenib is potential therapeutic medicine for nervous system disease. In this study, we preliminarily studied the possible mechanism of dabrafenib in the treatment of multiple sclerosis from the perspective of ferroptosis. First, we observed that dabrafenib significantly improved symptoms of gait abnormalities, limb weakness or paralysis, and down-regulated levels of spinal cord inflammation in an experimental autoimmune encephalitis (EAE) model. Meanwhile, we also observed that dabrafenib could inhibit the proteins of ferroptosis in spinal cord tissue of EAE mice by Western blot. The results of immunohistochemical analysis showed that the effect of dabrafenib on ferroptosis mainly occurred in microglia. Second, dabrafenib was demonstrated to be able to inhibit the S phase of the cell cycle, reduce ROS levels, and reinstate mitochondrial activity in the LPS-induced BV2 inflammatory cell model. Futhermore, we found that dabrafenib inhibits P-JAK2 and P-STAT3 activation by acting Axl receptor, which in turn prevents neurogenic inflammation in microglia. The co-stimulated BV2 cell model with LPS and Erastin also verified these findings. Ultimately, the Axl knockout mice used to construct the EAE model allowed for the confirmation that dabrafenib prevented ferroptosis in microglia by up-regulating Axl receptor, which reduced the inflammatory demyelination associated with EAE. In summary, our research demonstrates the advantages of dabrafenib in multiple sclerosis treatment, which can prevent ferroptosis in microglia in multiple sclerosis through up-regulating Axl receptor, thus halting the progression of multiple sclerosis.

MAP kinase inhibitor PD98059 regulates Th1, Th9, Th17, and natural T regulatory cells in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis

Experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS), provides significant insights into the mechanisms that initiate and drive autoimmunity. MS is a chronic autoimmune disease of the central nervous system, characterized by inflammatory infiltration associated with demyelination. T lymphocyte cells play a crucial role in MS, whereas natural T regulatory (nTreg) cells prevent autoimmune inflammation by suppressing lymphocyte activity. This study sought to investigate the role of PD98059, a selective MAP kinase inhibitor, in Th1, Th9, Th17, and nTreg cells using the SJL/J mouse model of EAE. Following EAE development, the mice were intraperitoneally administered PD98059 (5 mg/kg for two weeks) daily. We evaluated the effects of PD98059 on Th1 (IFN-γ and T-bet), Th9 (IL-9 and IRF4), Th17 (IL-17A and RORγT), and nTreg (FoxP3 and Helios) cells in the spleen using flow cytometry. Moreover, we explored the effects of PD98059 on the IFN-γ, T-bet, IL-9, IRF4, IL-17A, RORγT, FoxP3, and Helios mRNA and protein levels in brain tissues using qRT-PCR and Western blot analyses. PD98059 treatment significantly decreased the proportion of CD4+IFN-γ+, CD4+T-bet+, CD4+IL-9+, CD4+IRF4+, CD4+IL-17A+, CD4+RORγT+, CD4+IL-17A+, and CD4+RORγT+ cells while increasing that of CD4+FoxP3+ and CD4+Helios+ cells. In addition, PD98059 administration decreased the mRNA and protein levels of IFN-γ, T-bet, IL-9, IRF4, IL-17A, and RORγT but increased those of FoxP3 and Helios in the brain tissue of EAE mice. Our findings suggest that PD98059 corrects immune dysfunction in EAE mice, which is concurrent with the modulation of multiple signaling pathways.

Tolerogenic Lipid Nanoparticles for Delivering Self-Antigen mRNA for the Treatment of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis is a disease caused by autoantigen-responsive immune cells that disrupt the myelin in the central nervous system (CNS). Although immunosuppressive drugs are used to suppress symptoms, no definitive therapy exists. As in the experimental autoimmune encephalitis (EAE) model of multiple sclerosis, a partial sequence of the myelin oligodendrocyte glycoprotein (MOG35-55) was identified as a causative autoantigen. This suggests that the induction of immune tolerance that is specific to MOG35-55 would be a fundamental treatment for EAE. We previously reported that lipid nanoparticles (LNPs) containing an anionic phospholipid, phosphatidylserine (PS), in their lipid composition, can be used to deliver mRNA and that this leads to proteins of interest to be expressed in the spleen. In addition to the targeting capability of PS, PS molecules avoid activating the immune system. Physiologically, the recognition of PS on apoptotic cells suppresses immune activation against these cells by releasing cytokines, such as interleukin-10 (IL-10) and transforming growth factor (TGF)-β that negatively regulate immunity. In this study, we tested whether mRNA delivery of autoantigens to the spleen by PS-LNPs causes the expression of MOG35-55 antigens with minimal immune stimulation and whether this could be used to treat an EAE model by inducing immune tolerance.

Tr1 cell-mediated protection against autoimmune disease by intranasal administration of a fusion protein targeting cDC1 cells

Curative therapies against autoimmune diseases are lacking. Indeed, most of the currently available treatments are only targeting symptoms. We have developed a novel strategy for a therapeutic vaccine against autoimmune diseases based on intranasal administration of a fusion protein tolerogen, which consists of a mutant, enzymatically inactive, cholera toxin A1 (CTA1)-subunit genetically fused to disease-relevant high-affinity peptides and a dimer of D-fragments from protein A (DD). The CTA1 R7K mutant - myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein (PLP) - DD (CTA1R7K-MOG/PLP-DD) fusion proteins effectively reduced clinical symptoms in the experimental autoimmune encephalitis model of multiple sclerosis. The treatment induced Tr1 cells, in the draining lymph node, which produced interleukin (IL)-10 and suppressed effector clusters of differentiation 4+ T-cell responses. This effect was dependent on IL-27 signaling because treatment was ineffective in bone marrow chimeras lacking IL-27Ra within their hematopoietic compartment. Single-cell RNA sequencing of dendritic cells in draining lymph nodes demonstrated distinct gene transcriptional changes of classic dendritic cells 1, including enhanced lipid metabolic pathways, induced by the tolerogenic fusion protein. Thus, our results with the tolerogenic fusion protein demonstrate the possibility to vaccinate and protect against disease progression by reinstating tolerance in multiple sclerosis and other autoimmune diseases.

Myelin-associated glycoprotein activation triggers glutamate uptake by oligodendrocytes in vitro and contributes to ameliorate glutamate-mediated toxicity in vivo.

Myelin-associated glycoprotein (MAG) is a key molecule involved in the nurturing effect of myelin on ensheathed axons. MAG also inhibits axon outgrowth after injury. In preclinical stroke models, administration of a function-blocking anti-MAG monoclonal antibody (mAb) aimed to improve axon regeneration demonstrated reduced lesion volumes and a rapid clinical improvement, suggesting a mechanism of immediate neuroprotection rather than enhanced axon regeneration. In addition, it has been reported that antibody-mediated crosslinking of MAG can protect oligodendrocytes (OLs) against glutamate (Glu) overload by unknown mechanisms. To unravel the molecular mechanisms underlying the protective effect of anti-MAG therapy with a focus on neuroprotection against Glu toxicity. MAG activation (via antibody crosslinking) triggered the clearance of extracellular Glu by its uptake into OLs via high affinity excitatory amino acid transporters. This resulted not only in protection of OLs but also nearby neurons. MAG activation led to a PKC-dependent activation of factor Nrf2 (nuclear-erythroid related factor-2) leading to antioxidant responses including increased mRNA expression of metabolic enzymes from the glutathione biosynthetic pathway and the regulatory chain of cystine/Glu antiporter system xc- increasing reduced glutathione (GSH), the main antioxidant in cells. The efficacy of early anti-MAG mAb administration was demonstrated in a preclinical model of excitotoxicity induced by intrastriatal Glu administration and extended to a model of Experimental Autoimmune Encephalitis showing axonal damage secondary to demyelination. MAG activation triggers Glu uptake into OLs under conditions of Glu overload and induces a robust protective antioxidant response.

Perfusion with 10% neutral-buffered formalin is equivalent to 4% paraformaldehyde for histopathology and immunohistochemistry in a mouse model of experimental autoimmune encephalomyelitis.

Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139-151 subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice (P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.

Liraglutide attenuate central nervous inflammation and demyelination through AMPK and pyroptosis‐related NLRP3 pathway

AimsMultiple sclerosis (MS) still maintains increasing prevalence and poor prognosis, while glucagon‐like peptide‐1 receptor (GLP‐1R) agonists show excellent neuroprotective capacities recently. Thus, we aim to evaluate whether the GLP‐1R agonist liraglutide (Lira) could ameliorate central nervous system demyelination and inflammation.MethodsThe therapeutic effect of Lira was tested on experimental autoimmune encephalitis (EAE) in vivo and a microglia cell line BV2 in vitro.ResultsLira administration could ameliorate the disease score of EAE mice, delay the disease onset, ameliorate pathological demyelination and inflammation score in lumbar spinal cord, reduce pathogenic T helper cell transcription in spleen, restore phosphorylated adenosine monophosphate‐activated protein kinase (pAMPK) level, autophagy level, and inhibit pyroptosis‐related NLR family, pyrin domain‐containing protein 3 (NLRP3) pathway in lumbar spinal cord. Additionally, cell viability test, lactate dehydrogenase release test, and dead/live cell staining test for BV2 cells showed Lira could not salvage BV2 from nigericin‐induced pyroptosis significantly.ConclusionLira has anti‐inflammation and anti‐demyelination effect on EAE mice, and the protective effect of Lira in the EAE model may be related to regulation of pAMPK pathway, autophagy, and NLRP3 pathway. However, Lira treatment cannot significantly inhibit pyroptosis of BV2 cells in vitro. Our study provides Lira as a potential candidate for Multiple Sclerosis treatment.

Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques

Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression.The authors describe quantitatively and qualitatively different forms of experimental autoimmune encephalitis (EAE) in cynomolgus macaques. They found that bacterial contaminants within recombinant human myelin oligodendrocyte glycoprotein seemed to aggravate disease evolution. They provide anatomopathological features of fulminant and progressive forms of EAE, allowing them to distinguish specific factors influencing the evolution of this model of autoimmune demyelinating disease.

Assessment of Intravascular Perfusion with 10% Neutral Buffered Formalin Versus 4% Paraformaldehyde for Histopathology and Immunohistochemistry in a Mouse Model of Experimental Autoimmune Encephalitis

Intravascular (IV) perfusion of tissue fixative is a technique that is commonly utilized in the field of neuroscience as central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream morphologic readouts. In the literature, IV perfusion with 4% paraformaldehyde (PFA) is most referenced, although 10% neutral buffered formalin (NBF) is more commonly used for immersion fixation in both clinical and experimental pathology laboratories and has also been used in perfusion fixation. Unlike 4% PFA, 10% NBF is stable at room temperature and is not light sensitive. The study objective was to compare the frequency and severity of handling and fixation artifacts (including tissue tears, folds, oligodendrocyte halos, perivascular clefts, vacuolation, and dark neuron artifacts), semi-quantitative inflammation and necrosis scores, and quantitative immunohistochemistry following terminal IV perfusion of mice with 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). We hypothesized that no differences would be observed between the two fixatives after IV perfusion. SJL mice were immunized with 50µg of PLP139–151 subcutaneously and at day 20 mice were harvested for tissue collection. A total of 24 mice were used; 12 were control animals not immunized (4 perfused with 10% NBF, 4 perfused with 4% PFA and 4 were not perfused, but brain and spinal cord were collected fresh and then immersion fixed in 10% NBF). An additional 12 were immunized and fixed in the same fashion. Histologically, spinal cord sections from PFA perfused mice had less severe tissue folds than those from NBF perfused mice (P<0.01). NBF perfused mice had less severe dark neuron artifact than PFA perfused mice (P<0.001). Immersion fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion fixed animals. No significant differences were noted in EAE histopathology scores for inflammation, demyelination, or necrosis in immunized animals regardless of the method of fixation. Finally, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed regardless of method of fixation, although scores for EAE animals were significantly higher than controls. These findings indicate that whole body perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques for the study of neuropathology, especially in an EAE model.

Astrocyte TAK1-mediated production of chemotactic cues are necessary for the localization of CD4+T cell in gray matter in an EAE model with severe neurodegeneration.

Abstract Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system that is driven by peripheral immune cell infiltration and glial cell activation. The pathological hallmark of MS is primary demyelination, but recent evidence suggests gray matter neuronal damage is a major component of the irreversible nature of the disease. While T cells are found in both gray and white matter of MS tissue, they are typically confined to the white matter of the most commonly used mouse model of MS, experimental autoimmune encephalitis (EAE). Thus, genesis of T cells in the gray matter during MS pathogenesis is poorly understood. In this study, we used a modified EAE model (Type-B EAE) that displays severe neuronal damage without disease remission to investigate the role of T cells in neurodegeneration. Using immunohistochemistry and confocal imaging, we show that CD4+ T cells migrate to the spinal cord gray matter with selective preference for the ventral horns. Compared to CD4+ T cells in white matter, gray matter-infiltrated CD4+ T cells were mostly immobile (indicative of cell–cell interactions) and can be seen interacting directly with neuronal bodies. T cell-specific deletion of CXCR2 significantly decreased CD4+ T cell infiltration into gray matter in Type-B EAE mice. Inhibition of astrocyte chemokine production such as CXCL1, which was heightened in gray matter of Type-B EAE mice, by astrocyte-specific deletion of TAK1 successfully prevented T cell migration into spinal cord gray matter and rescued neuronal damage and motor dysfunction in Type-B EAE mice. This is the first report report that identifies the mechanism by which encephalomyelitic CD4+ T cells migrate to spinal cord gray matter, which correlates with neuronal damage.

The Contribution of Connexin 43 in Choroid Plexus to Experimental Autoimmune Encephalomyelitis

Abstract Multiple Sclerosis (MS) is the most common progressive neurologic disease among young adults worldwide; yet the pathogenesis remains poorly understood. The myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalitis (EAE) in mice is a robust model and has been extensively used to understand the pathophysiology of MS. The expression of connexin 43 (CX43), a gap junction protein, has been shown to be enhanced in the choroid plexus during the development of acute EAE, with significantly increased CX43 expression at the peak of the disease. However, the contribution of such increased CX43 expression to the development of EAE has yet to be fully characterized. Using the Cre-Lox platform, we generated mice with conditional deletion of CX43 in the ciliated columnar epithelium of the choroid plexus (Foxj1Cre-CX43 KO mice; referred to as CX43 KO mice hereafter). We employed the MOG-induced EAE model in both male and female CX43 KO mice to understand the role of CX43 in the pathogenesis of EAE. Male CX43 and WT mice displayed comparable peak EAE clinical disease scores of 1.83±0.33 and 1.75±0.66, respectively. Female CX43 KO, however, displayed a significantly reduced peak EAE clinical disease score of 0.33±0.24 compared to 1.15±0.39 in female WT animals. These results suggest a role for CX43 in the ciliated columnar epithelium of choroid plexus to the development of EAE in female, not male, mice and underscore the need for further exploration of mechanism(s) underpinning the role of CX43 in the pathogenesis of EAE.

Regulatory Dendritic Cells Induced by K313 Display Anti-Inflammatory Properties and Ameliorate Experimental Autoimmune Encephalitis in Mice.

As a GSK-3β inhibitor reported by our group, K313 is a novel benzoxazole derivative and displays anti-inflammatory properties in RAW264.7 macrophages without cytotoxicity. The activity of GSK-3β affects the differentiation and maturation of bone marrow-derived dendritic cells (DCs). This study aims to investigate whether K313 can be used to induce regulatory/tolerogenic dendritic cells (DCregs), and the therapeutic effects of DCregs induced by K313 in the autoimmune model of experimental autoimmune encephalitis (EAE). The results show that compared with LPS stimulated mature DCs, K313-treated bone marrow-derived DCs display obvious tolerogenic characteristics with decreased expression of co-stimulatory molecules, downregulated secretions of pro-inflammatory cytokines and unregulated secretion of anti-inflammatory cytokine IL-10. The above characteristics conform to the typical phenotypes of DCregs. Moreover, K313-modified DCregs inhibit antigen-specific T cell responses in vitro. Furthermore, by adoptive transfer, K313 modified DCregs to the EAE mice, and the development of disease was ameliorated to some extent. In addition, treatment with K313-modified DCregs also significantly reduced the percentages of splenetic Th1 and Th17 cells and increased the percentage of regulatory T cells in EAE mice. In conclusion, K313-modified DCregs show anti-inflammatory properties in vitro and have a significant positive effect on the EAE disease in vivo. Our data indicate that K313-induced DCregs pulsed with auto-antigen might have potential use as a therapeutic approach for autoimmune inflammation of the central nervous system.

Evaluation of Myelin Radiotracers in the Lysolecithin Rat Model of Focal Demyelination: Beware of Pitfalls!

The observation that amyloid radiotracers developed for Alzheimer's disease bind to cerebral white matter paved the road to nuclear imaging of myelin in multiple sclerosis. The lysolecithin (lysophosphatidylcholine (LPC)) rat model of demyelination proved useful in evaluating and comparing candidate radiotracers to target myelin. Focal demyelination following stereotaxic LPC injection is larger than lesions observed in experimental autoimmune encephalitis models and is followed by spontaneous progressive remyelination. Moreover, the contralateral hemisphere may serve as an internal control in a given animal. However, demyelination can be accompanied by concurrent focal necrosis and/or adjacent ventricle dilation. The influence of these side effects on imaging findings has never been carefully assessed. The present study describes an optimization of the LPC model and highlights the use of MRI for controlling the variability and pitfalls of the model. The prototypical amyloid radiotracer [11C]PIB was used to show that in vivo PET does not provide sufficient sensitivity to reliably track myelin changes and may be sensitive to LPC side effects instead of demyelination as such. Ex vivo autoradiography with a fluorine radiotracer should be preferred, to adequately evaluate and compare radiotracers for the assessment of myelin content.

3389 Coagulation Factor Xll-mediated contact system and its role in adaptive and innate immune cell movement

OBJECTIVES/SPECIFIC AIMS: The objectives of this study are to 1) expand upon the paradigm of HK-D5 accelerated immune cell chemotaxis; 2) characterize the role of FXII in murine models of immune-mediated disease using FXII KO mice and a small molecule inhibitor of FXIIa. METHODS/STUDY POPULATION: To test whether the addition of HK-D5 peptide would accelerate C-C chemokine receptor type 2 (CCR2)-mediated chemotaxis in vitro, a real-time transwell chemotaxis assay was developed utilizing human THP-1 monocyte cell line (Fig 1). For in vivo studies, both pharmacologic FXIIa antagonism and FXll KO mice were used. Genotyping, histopathological review, FXll protein expression, and active partial thromboplastin time (aPTT) measurements were used to phenotypically characterize FXII KO mice compared to C57BL/6 wild type mice (Fig 2). Subsequently, the FXll KO mice were studied in varied models of immune-mediated disease, including LPS-induced sepsis and experimental autoimmune encephalitis (EAE), to see if and how the absence of FXll can mitigate disease severity. The EAE model involved active immunization with myelin oligodendrocyte glycoprotein (MOG) and measurement of established clinical disease severity scores. The LPS sepsis model involved an intraperitoneal injection of LPS followed by 48-hour monitoring of core body temperature using subdermal temperature transponders as a proxy for inflammatory events related to septic shock (Fig 3). RESULTS/ANTICIPATED RESULTS: HK-D5 significantly accelerates CCR2-mediated chemotaxis compared to chemokine alone (p = 0.001) similar to HK-D5’s ability to accelerate CCR7-mediated chemotaxis as previously established. The FXll KO mice were backcrossed to the C57BL/6J background and confirmed by genotyping and complete absence of FXII protein in plasma. Compared to the control, FXII KO mice have a significantly prolonged aPTT without evidence of bleeding abnormalities, which confirms the expected phenotype previously described and recapitulates what is observed in Factor XII deficiency in humans. KO mice showed no significant gross or histopathological differences in secondary lymphoid structures compared to the control. Immunohistochemistry confirmed well-organized lymphoid structures with intact B- and T-cell populations. FXll KO mice are protected in LPS-induced septic shock and EAE models. Regarding the EAE model, FXIIa inhibition significantly reduced disease severity compared to control. In the LPS model, FXll KO mice recover within 24 hours after LPS-challenge measured subjectively and objectively by core body temperature measurement. DISCUSSION/SIGNIFICANCE OF IMPACT: The current study and previous findings suggest a novel immune signaling mechanism by which a peptide fragment of high molecular-weight kininogen (HK-D5) acts as an accelerant of both innate and adaptive immune cell chemotaxis in multiple immune contexts. This has broad implications regarding a mechanism of immune-mediated inflammation in a variety of disease states, which might be amenable to the targeting this pathway for therapeutic intent.

TFL Deletion Induces Incredible CXCL13 Secretion and Cachexia in Vavp-Bcl2Transgenic Mice

Diffuse large B-cell lymphomas (DLBCL) are heterogeneous diseases caused by several genetic aberrations. The novel post-transcriptional regulator gene called transformed follicular lymphoma (TFL) was first identified from t(2;6)(p12;q23), which appeared during the transformation of FL to DLBCL (Minagawa et al. Br J Haematol 2007). Normal human lymphocytes generally express TFL, but it is defective in some leukemia/lymphoma cell lines. TFL overexpression in such cell lines inhibited cell growth, suggesting that TFL functions as a tumor suppressor (Minagawa et al. Mol Cancer Res 2009). TFL locates in mRNA processing body in the cytoplasm and has the unique CCCH-type zinc finger motif functioning as RNase. TFL regulates several cytokines, including IL-2, IL-6, IL-10, TNF-α, and IL-17a, via mRNA degradation. In an experimental autoimmune encephalitis model, TFL null mice (TFL-/-) demonstrated persistent paralysis, resulting from more infiltration of Th17 cells into CNS with markedly increased IL-17a mRNA levels. Therefore, a TFL-driven feedback mechanism for excessive inflammation is indispensable to suppress T-cell-mediated autoimmune diseases (Minagawa et al. J Immunol 2014).TFL deletion examined by FISH using a 110kbp DNA probe containing an entireTFL locus was found in 12.8% of mature B-cell lymphomas (n=86, FL=30, DLBCL=40). However, the pathological significance of TFL deletion has not yet been clarified. To investigate how TFL loss affects lymphoma biology, we developed VavP-bcl2 transgenic (Bcl2-Tg)/TFL-/-mice. Although the survival of TFL-/- was comparable to the wild-type, Bcl2-Tg/TFL-/- died about 19 weeks earlier than Bcl2-Tg (Fig. 1). Both strains developed lymphadenopathy and splenomegaly similarly. No different microscopic finding was noted in lymph nodes, spleen, or bone marrow (BM). No additional malignancy was found in Bcl2-Tg/TFL-/- on autopsy. However, significant body weight loss appeared by 30 weeks in Bcl2-Tg/TFL-/- but not in Bcl2-Tg (Fig. 3).To identify what causes earlier death in Bcl2-Tg/TFL-/-, we carefully examined the phenotypic change of BM lymphocytes. We found a unique B220-IgM+ population in Bcl2-Tg BM, which was not found in wild-type. We speculated that TFL deficiency in this population might drive the deterioration in Bcl2-Tg/TFL-/-. To identify which mRNA was dysregulated by TFL deficiency, we comprehensively analyzed mRNA expression profiles in B220-IgM+ cells in both strains using cDNA microarray chip. Among several genes upregulated at least threefold in Bcl2-Tg/TFL-/- than Bcl2-Tg, we paid attention to CXCL13, the mRNA expression of which in Bcl2-Tg/TFL-/- was 4.19-fold higher than that in Bcl2-Tg (p=0.03). In fact, CXCL13 concentration in BM extracellular fluid as well as plasma in Bcl2-Tg/TFL-/- showed incredible increase in a logarithmic scale (Fig. 2). As a noteworthy event, body weight loss in Bcl2-Tg/TFL-/- followed the increase of CXCL13 in plasma by 30 weeks (Fig. 3). To confirm that TFL post-transcriptionally regulates CXCL13 mRNA through the degradation of its 3′UTR, we performed a reporter assay with a plasmid vector containing 3′UTR of CXCL13 mRNA. Co-transfection with a TFL expression vector showed decreased luciferase activity compared to the control. This suggests that TFL directly regulates CXCL13 mRNA via its 3′UTR degradation. This regulation occurs more prominently in B-cell lineage rather than myeloid or T-cell lineage, whereas IL-2 mRNA regulation occurs promiscuously.CXCL13 secretion was significantly increased in the culture supernatant of BM cells but not spleen cells derived from Bcl2-Tg/TFL-/-. We further sorted several cell populations, including B220-IgM+ in BM, and cultured them for 96 h. CXCL13 secretion from B220-IgM+ population was increased significantly compared to other populations. Thus, we concluded that B220-IgM+ cells in BM are the main producer of CXCL13 in Bcl2-Tg/TFL-/-. Loss of TFL-driven attenuation for excessive inflammation in lymphoma-bearing mice could contribute to the short survival. It is of interest whether high plasma CXCL13 directly affects cachexia and early death in Bcl2-Tg/TFL-/-.TFL deletion in human lymphoma might contribute not only to malignant transformation but also to a major B symptom, i.e., weight loss. Our findings may open a new window for the predictive factor on the prognosis of B-cell lymphoma and/or new therapeutic intervention by targeting CXCL13. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inactivation of sphingosine-1-phosphate receptor 2 (S1PR2) decreases demyelination and enhances remyelination in animal models of multiple sclerosis

Multiple sclerosis is an inflammatory disease of the central nervous system (CNS) in which multiple sites of blood-brain barrier (BBB) disruption, focal inflammation, demyelination and tissue destruction are the hallmarks. Here we show that sphingosine-1-phosphate receptor 2 (S1PR2) has a negative role in myelin repair as well as an important role in demyelination by modulating BBB permeability. In lysolecithin-induced demyelination of adult mouse spinal cord, S1PR2 inactivation by either the pharmacological inhibitor JTE-013 or S1PR2 gene knockout led to enhanced myelin repair as determined by higher numbers of differentiated oligodendrocytes and increased numbers of remyelinated axons at the lesion sites. S1PR2 inactivation in lysolecithin-induced demyelination of the optic chiasm, enhanced oligodendrogenesis and improved the behavioral outcome in an optokinetic reflex test. In order to see the effect of S1PR2 inactivation on demyelination, experimental autoimmune encephalitis (EAE) was induced by MOG-peptide. S1PR2 inhibition or knockout decreased the extent of demyelinated areas as well as the clinical disability in this EAE model. Both toxin induced and EAE models showed decreased BBB leakage and reduced numbers of Iba1+ macrophages following S1PR2 inactivation. Our results suggest that S1PR2 activity impairs remyelination and also enhances BBB leakage and demyelination. The former effect could be mediated by Nogo-A, as antagonism of this factor enhances remyelination and S1PR2 can act as a Nogo-A receptor.

Multiple sclerosis pathogenesis: missing pieces of an old puzzle.

Traditionally, multiple sclerosis (MS) was considered to be a CD4 T cell-mediated CNS autoimmunity, compatible with experimental autoimmune encephalitis model, which can be characterized by focal lesions in the white matter. However, studies of recent decades revealed several missing pieces of MS puzzle and showed that MS pathogenesis is more complex than the traditional view and may include the following: a primary degenerative process (e.g. oligodendroglial pathology), generalized abnormality of normal-appearing brain tissue, pronounced gray matter pathology, involvement of innate immunity, and CD8 T cells and B cells. Here, we review these findings and discuss their implications in MS pathogenesis.

Discovery of a novel human anti-IL-2 antibody that potentiates Regulatory T cells by a structure-based mechanism

Abstract Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Treg). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Treg cells. We report the generation of the first fully human anti-IL-2 antibody, F5111.2 that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalitis, and protected mice against xenogeneic Graft-versus-Host-Disease (GvHD). These results suggest that IL-2-F5111.2 may provide a new immunotherapy to treat autoimmune diseases and GvHD.

Human antibodies against the myelin oligodendrocyte glycoprotein can cause complement-dependent demyelination

BackgroundAntibodies to the myelin oligodendrocyte glycoprotein (MOG) are associated with a subset of inflammatory demyelinating diseases of the central nervous system such as acute disseminated encephalomyelitis and neuromyelitis optica spectrum disorders. However, whether human MOG antibodies are pathogenic or an epiphenomenon is still not completely clear. Although MOG is highly conserved within mammals, previous findings showed that not all human MOG antibodies bind to rodent MOG. We therefore hypothesized that human MOG antibody-mediated pathology in animal models may only be evident using species-specific MOG antibodies.MethodsWe screened 80 human MOG antibody-positive samples for their reactivity to mouse and rat MOG using either a live cell-based assay or immunohistochemistry on murine, rat, and human brain tissue. Selected samples reactive to either human MOG or rodent MOG were subsequently tested for their ability to induce complement-mediated damage in murine organotypic brain slices or enhance demyelination in an experimental autoimmune encephalitis (EAE) model in Lewis rats. The MOG monoclonal antibody 8-18-C5 was used as a positive control.ResultsOverall, we found that only a subset of human MOG antibodies are reactive to mouse (48/80, 60%) or rat (14/80, 18%) MOG. Purified serum antibodies from 10 human MOG antibody-positive patients (8/10 reactive to mouse MOG, 6/10 reactive to rat MOG), 3 human MOG-negative patients, and 3 healthy controls were tested on murine organotypic brain slices. Purified IgG from one patient with high titers of anti-human, mouse, and rat MOG antibodies and robust binding to myelin tissue produced significant, complement-mediated myelin loss in organotypic brain slices, but not in the EAE model. Monoclonal 8-18-C5 MOG antibody caused complement-mediated demyelination in both the organotypic brain slice model and in EAE.ConclusionThis study shows that a subset of human MOG antibodies can induce complement-dependent pathogenic effects in a murine ex vivo animal model. Moreover, a high titer of species-specific MOG antibodies may be critical for demyelinating effects in mouse and rat animal models. Therefore, both the reactivity and titer of human MOG antibodies must be considered for future pathogenicity studies.