Kinesin-5 inhibition improves neural regeneration in experimental autoimmune neuritis.

BACKGROUND: Autoimmune neuropathies can result in long-term disability and incomplete recovery, despite adequate first-line therapy. Kinesin-5 inhibition was shown to accelerate neurite outgrowth in different preclinical studies. Here, we evaluated the potential neuro-regenerative effects of the small molecule kinesin-5 inhibitor monastrol in a rodent model of acute autoimmune neuropathies, experimental autoimmune neuritis. METHODS: Experimental autoimmune neuritis was induced in Lewis rats with the neurogenic P2-peptide. At the beginning of the recovery phase at day 18, the animals were treated with 1 mg/kg monastrol or sham and observed until day 30 post-immunisation. Electrophysiological and histological analysis for markers of inflammation and remyelination of the sciatic nerve were performed. Neuromuscular junctions of the tibialis anterior muscles were analysed for reinnervation. We further treated human induced pluripotent stem cells-derived secondary motor neurons with monastrol in different concentrations and performed a neurite outgrowth assay. RESULTS: Treatment with monastrol enhanced functional and histological recovery in experimental autoimmune neuritis. Motor nerve conduction velocity at day 30 in the treated animals was comparable to pre-neuritis values. Monastrol-treated animals showed partially reinnervated or intact neuromuscular junctions. A significant and dose-dependent accelerated neurite outgrowth was observed after kinesin-5 inhibition as a possible mode of action. CONCLUSION: Pharmacological kinesin-5 inhibition improves the functional outcome in experimental autoimmune neuritis through accelerated motor neurite outgrowth and histological recovery. This approach could be of interest to improve the outcome of autoimmune neuropathy patients.

Lactobacillus paracasei L9 affects disease progression in experimental autoimmune neuritis by regulating intestinal flora structure and arginine metabolism.

BACKGROUND: Autoimmune neuropathies are common peripheral nervous system (PNS) disorders. Environmental influences and dietary components are known to affect the course of autoimmune diseases. Intestinal microorganisms can be dynamically regulated through diet, and this study combines intestinal microorganisms with diseases to open up new therapeutic ideas. METHODS: In Lewis rats, a model of EAN was established with P0 peptide, Lactobacillus were used as treatment, serum T-cell ratio, inflammatory factors, sciatic neuropathological changes, and pathological inflammatory effects on intestinal mucosa were detected, and fecal metabolomics and 16 s microbiome analysis were performed to further explore the mechanism. RESULTS: In the EAN rat model, Lactobacillus paracasei L9 (LP) could dynamically regulate the CD4+/CD8+T balance in serum, reduce serum IL-1, IL-6 and TNF-α expression levels, improve sciatic nerve demyelination and inflammatory infiltration, and reduce nervous system score. In the rat model of EAN, intestinal mucosa was damaged. Occludin and ZO-1 were downregulated. IL-1, TNF-α and Reg3γ were upregulated. LP gavage induced intestinal mucosa recovery; occludin and ZO-1 upregulation; IL-1, TNF-α and Reg3γ downregulation. Finally, metabolomics and 16 s microbiome analysis were performed, and differential metabolites were enriched with an important metabolic pathway, arginine and proline metabolism. CONCLUSION: LP improved EAN in rats by influencing intestinal community and the lysine and proline metabolism.

[Expression of glycolytic genes in immune cells and changes of related immune cells in experimental autoimmune neuritis].

Objective: To investigate the expression of glycolytic genes in immune cells and the changes of related immune cells in experimental autoimmune neuritis (EAN), and deepen the understanding of pathogenesis of EAN. Methods: Twenty-four male C57BL/6 mice (6-8 weeks old, 18-20 g) were divided into four groups according to the random number table method: control group (P0180-199 was replaced by PBS during modeling and mice were sacrificed on the 16th day), EAN mice were sacrificed on the 8th day after the end of modeling (EAN 8 d), EAN mice were sacrificed on the 16th day after the end of modeling (EAN 16 d), and EAN mice received drug intervention and were sacrificed on the 16th day after the end of modeling (2-DG was intraperitoneally injected since the day of the first immunization, 550 mg/kg; EAN 16 d+2-DG), with 6 rats in each group. The clinical symptoms and clinical scores were observed and recorded daily. At the end of the experiment, the mice were sacrificed under chloral hydrate anesthesia, and the serum, spleen, sciatic nerve and other tissues of each group were collected. The degree of inflammatory cell infiltration and demyelination of sciatic nerve were observed by hematoxylin and eosin (HE) staining and luxol fast blue (LFB) staining. Flow cytometry was used to detect the proportion of M1 macrophages, Th17 cells and Tregs cells. The mRNA expression levels of glycolysis-related genes (mTORC1, HIF1α, GLUT1 and LDHA) were detected by RT-PCR. Western blotting was used to detect the level of pan-lysine lactate in macrophages and sciatic nerve tissue. Results: The expression of glycolysis-related genes (mTORC1, HIF1α, GLUT1 and LDHA) in spleen M1 macrophages and sciatic nerve was significantly up-regulated in EAN 16 d group, compared with control, EAN 8 d and EAN 16 d+2-DG groups (all P<0.05). The relative pan-lysine lactate (pankla) expression level of spleen M1 macrophages (1.25±0.02) and sciatic nerve tissue (1.23±0.26) significantly increased in EAN 16 d group, compared with control, EAN 8 d and EAN 16 d+2-DG groups (M1 macrophages: 0.12±0.10, 1.07±0.12 and 0.42±0.07; sciatic nerve: 0.10±0.12, 0.87±0.20 and 0.36±0.05) (all P<0.05). The expression of glycolytic genes in splenic CD4+T cells showed an increasing trend, but there were no statistically significant differences among the groups, and the expression of glycolytic genes did not decrease significantly after 2-DG treatment (all P>0.05). The proportion of spleen M1 macrophages in the control group, EAN 8 d group, EAN 16 d group and EAN 16 d+2-DG group was 4.28±0.13, 7.54±0.25, 13.16±0.33 and 4.13±0.38 respectively, which was significantly higher in the EAN 16 d group (all P<0.05). The proportion of spleen Th17 cells in the four groups was 3.78±0.03, 8.24±0.55, 12.30±1.34 and 4.83±0.01, respectively, which was significantly higher in the EAN 16 d group (all P<0.05). The proportion of spleen Tregs cells in the four groups was 10.01±1.05, 7.54±0.70, 3.82±0.47 and 8.22±1.21, respectively, which was significantly lower in the EAN 16 d group (all P<0.05). Conclusions: The expression of glycolytic genes in splenic macrophages significantly increases during EAN, but not in CD4+T cells. The proportion of M1 macrophages and Th17 cells in spleen gradually increases, while the proportion of Tregs cells gradually decreases.

CX3CR1 But Not CCR2 Expression Is Required for the Development of Autoimmune Peripheral Neuropathy in Mice.

One hallmark of Guillain-Barre syndrome (GBS), a prototypic autoimmune peripheral neuropathy (APN) is infiltration of leukocytes (macrophages and T cells) into peripheral nerves, where chemokines and their receptors play major roles. In this study, we aimed to understand the potential contribution of chemokine receptors CCR2 and CX3CR1 in APN by using a well-established mouse model, B7.2 transgenic (L31) mice, which possesses a predisposed inflammatory background. We crossbred respectively CCR2KO and CX3CR1KO mice with L31 mice. The disease was initiated by partial ligation on one of the sciatic nerves. APN pathology and neurological function were evaluated on the other non-ligated sciatic nerve/limb. Our results revealed that L31/CX3CR1KO but not L31/CCR2KO mice were resistant to APN. CX3CR1 is needed for maintaining circulating monocyte and CD8+ T cell survival. While migration of a significant number of activated CD8+ T cells to peripheral nerves is essential in autoimmune response in nerve, recruitment of monocytes into PNS seems optional. Disease onset is independent of CCR2 mediated blood-derived macrophage recruitment, which can be replaced by compensatory proliferation of resident macrophages in peripheral nerve. CX3CR1 could also contribute to APN via its critical involvement in maintaining nerve macrophage phagocytic ability. We conclude that blockade of CX3CR1 signaling may represent an interesting anti-inflammatory strategy to improve therapeutic management for GBS patients.

Protective Role of Limitrin in Experimental Autoimmune Optic Neuritis.

Purpose: This study investigated the role of limitrin in the pathogenesis of demyelinating optic neuritis using an experimental autoimmune optic neuritis (EAON) model. Methods: EAON was induced in mice via subcutaneous injection with myelin oligodendrocyte glycoprotein peptide. Limitrin protein and mRNA expression were examined in the optic nerve before and after EAON induction. Proinflammatory cytokine expression profiles and degree of glial activation were compared between wild-type (WT) and limitrin knockout mice by real-time PCR and histologic analysis, respectively, after EAON induction. Plasma limitrin levels in patients with optic neuritis and healthy controls were measured by ELISA. Results: Limitrin expression, observed in astrocytes in the optic nerve of WT mice, was lower in EAON-induced than in naïve WT mice. A comparative analysis of WT and limitrin knockout mice revealed that limitrin deficiency induced more severe neuroinflammation and glial hyperactivation in the optic nerve after EAON induction. Limitrin-deficient astrocytes were more chemotactically responsive to neuroinflammatory stimulation than WT astrocytes. Patients with optic neuritis demonstrated higher plasma limitrin levels than healthy controls (P = 0.0001), which was negatively correlated with visual acuity at the nadir of the optic neuritis attack (r = 0.46, P = 0.036). Conclusions: Limitrin deficiency induced severe neuroinflammation and reactive gliosis in the optic nerve after EAON induction. Our results imply that astrocyte-derived limitrin may protect against neuroinflammation by decreasing immune cell infiltration into the optic nerve. The plasma limitrin level may reflect the extent of blood-brain barrier disruption and provide a valuable biomarker reflecting the severity of optic neuritis.

T Cells from NOD-PerIg Mice Target Both Pancreatic and Neuronal Tissue.

It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D) pathogenesis. In fact, a large proportion of islet-infiltrating B lymphocytes in the NOD mouse model of T1D produce Abs directed against the neuronal type III intermediate filament protein peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B lymphocytes express the H and L chain Ig molecules from the intra-islet-derived anti-peripherin-reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development, and PerIg B lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T cells from a pool of naive precursors. We now report adoptively transferred T cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients but, depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4+ and CD8+ T cells. Ab depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8+ T cells but required CD4+ T cells. Surprisingly, sciatic nerve-infiltrating CD4+ cells had an expansion of IFN-γ- and TNF-α- double-negative cells compared with those within both islets and spleen. Nerve and islet-infiltrating CD4+ T cells also differed by expression patterns of CD95, PD-1, and Tim-3. Further studies found transitory early B lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice, allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B lymphocyte-dependent autoimmune neuritis.

M1 Macrophage Derived Exosomes Aggravate Experimental Autoimmune Neuritis via Modulating Th1 Response.

Guillain-Barré syndrome (GBS), an immune-mediated disorder affecting the peripheral nervous system, is the most common and severe acute paralytic neuropathy. GBS remains to be potentially life-threatening and disabling despite the increasing availability of current standard therapeutic regimens. Therefore, more targeted therapeutics are in urgent need. Macrophages have been implicated in both initiation and resolution of experimental autoimmune neuritis (EAN), the animal model of GBS, but the exact mechanisms remain to be elucidated. It has been increasingly appreciated that exosomes, a type of extracellular vesicles (EVs), are of importance for functions of macrophages. Nevertheless, the roles of macrophage derived exosomes in EAN/GBS remain unclear. Here we determined the effects of macrophage derived exosomes on the development of EAN in Lewis rats. M1 macrophage derived exosomes (M1 exosomes) were found to aggravate EAN via boosting Th1 and Th17 response, while M2 macrophage derived exosomes (M2 exosomes) showed potentials to mitigate disease severity via a mechanism bypassing Th1 and Th17 response. Besides, both M1 and M2 exosomes increased germinal center reactions in EAN. Further in vitro studies confirmed that M1 exosomes could directly promote IFN-γ production in T cells and M2 exosomes were not capable of inhibiting IFN-γ expression. Thus, our data identify a previously undescribed means that M1 macrophages amplify Th1 response via exosomes and provide novel insights into the crosstalk between macrophages and T cells as well.

Inflammatory oedema of nerve trunks may be pathogenic in very early Guillain-Barré syndrome.

The aim of this paper is to analyse the pathological background of very early Guillain-Barré (VEGBS) (≤ 4 days after onset) comparing it with initial stages of experimental autoimmune neuritis (EAN). The pathological hallmark of VEGBS is inflammatory oedema predominating in proximal nerve trunks. In EAN inflammatory oedema precedes the development of demyelination or axonal degeneration; such oedema may increase endoneurial fluid pressure (EFP) stretching the perineurium and constricting the transperineurial microcirculation. Centrofascicular or wedge-shaped areas of nerve ischemia have been reported in GBS and EAN. Additional support for proximal VEGBS pathology comes from electrophysiology showing alterations in late responses as the most frequent features, and ultrasonography illustrating that main changes rely on ventral rami of spinal nerves. Selective inefficiency of the blood-nerve barrier would explain the topography of changes in VEGBS. Increased serum neurofilament light chain concentration has recently been reported in VEGBS, with no difference between demyelinating and axonal subtypes. This is a marker of axonal damage, which could be correlated with endoneurial ischemia caused by increased EFP. Inflammatory oedema of proximal nerve trunks may be pathogenic in VEGBS, and consequently there is a pressing need for therapeutic strategies to stop its rapid impact on the axons.

Ultrastructural characterization of mitochondrial damage in experimental autoimmune neuritis.

Data are sparse about mitochondrial damage in GBS and in its most frequently employed animal model, experimental autoimmune neuritis (EAN). We here characterized changes in mitochondrial content and morphology at different time points during EAN by use of ultrastructural imaging and immunofluorescent labelling. Histological examination revealed that demyelinated axons and their adjacent Schwann cells showed reduced mitochondrial content and remaining mitochondria appeared swollen with greater diameter in Schwann cells and unmyelinated axons. Our findings indicate that in EAN, particularly mitochondria in Schwann cells are damaged. Further studies are warranted to address whether these changes are amenable to novel, mitoprotective treatments.

The dynamic expression of canonical Wnt/ß-catenin signalling pathway in the pathologic process of experimental autoimmune neuritis.

Background: Guillain-Barré syndrome (GBS), an autoimmune disease and an acute inflammation disorder, is currently the most frequent cause of acute flaccid paralysis worldwide. EAN, an animal model of GBS, is a CD4+ T cell-mediated autoimmune disease of the PNS. Wnt/ß-catenin signals are critically important to several fundamental aspects of peripheral nerve development and play a crucial role in Schwann cell proliferation. Here, we investigate the role of Wnt/ß-catenin signalling cascades in EAN rats.Methods: 28 male Lewis rats weighing 170 ± 10 g were randomly divided into control group (n = 7) and EAN groups (Early group; Peak group and Recovery group. n = 7 per group). EAN rats were immunized with P257-81 peptide; weighed daily, and the neurologic signs of EAN were evaluated every day. The sciatic nerve was taken on the days 10, 17, and 30 p.i. for H&E staining, transmission electron microscopy and immunohistochemical staining; blood samples were collected weekly from caudal vein to detect IFN-γ, IL-4, TGF-ß1; and the sciatic nerve was taken to examinate the dynamics expression of Wnt/ß-catenin pathway molecules.Results: In our study, we chose tail-root injection to better model GBS. Moreover, we observed that IFN-γ levels paralleled clinical EAN, and the levels of TGF-ß1 and IL-4 gradually increased and peaked in the recovery phase. In addition, we have shown that canonical Wnt signalling is upregulated and reached a peak in the late recovery phase.Conclusion: Our findings suggest that Wnt/ß-catenin signalling is associated with the promotion of remyelination in EAN rats.

Transcriptomes in rat sciatic nerves at different stages of experimental autoimmune neuritis determined by RNA sequencing.

Guillain-Barré syndrome (GBS) is characterized by acute immune-mediated peripheral neuropathy, which may result in rapidly progressive paralysis and fatal respiratory failure. As the underlying pathological mechanisms of GBS are unclear, we surveyed the transcriptome of rats with experimental autoimmune neuritis (EAN), a model of GBS. Briefly, sciatic nerves on both sides were collected from 8-10-week-old Lewis rats during early (10 days post-induction), peak (19 days) and late neuritis (30 days). Total RNA was sequenced to identify differentially expressed genes. Compared to control rats without induced neuritis, 33 genes were differentially expressed in the early phase (14 up-regulated and 19 down-regulated), with an adjusted P-value < 0·05 and |log2 fold-change| >1, as were 137 genes in the peak phase (126 up-regulated and 11 down-regulated) and 60 genes in the late phase (58 up-regulated and two down-regulated). Eleven of these genes were common to all stages, suggesting their crucial roles throughout the disease course. Analysis of protein-protein interactions revealed Fos, Ccl2, Itgax and C3 as node genes at different stages. Functional analysis of differentially expressed genes identified biological processes and pathways that are activated as neuritis progresses. This is the first genomewide gene expression study of peripheral nerves in experimental autoimmune neuritis model. Dynamic gene expression and significantly altered biological functions were detected in different phases of the disease, increasing our understanding of the molecular mechanisms underlying EAN and highlighting potential targets for its diagnosis and treatment.

Time Course of Axon and Myelin Degeneration in Peripheral Nerves in Experimental Autoimmune Neuritis Rats.

Experimental autoimmune neuritis (EAN) is an animal model for Guillain-Barré syndrome (GBS), which results in neurological symptoms and histopathological changes in peripheral nerves. In this model, the correlation between the progression of the disease and the histopathological changes is not clear. To further examine histopathological changes in peripheral nerves in EAN rats, sciatic nerves were sampled at onset (day 10), peak (day 16), and recovery (days 22 and 25) of neurological symptoms in P2(57-81)-peptide-administered rats. Axon and myelin degeneration was observed by light microscopy at onset, degeneration became severe at peak, and persisted at recovery. Densities of myelinated nerve fibers and myelin areas decreased from day 10 to a minimum on day 22. Slight axon and myelin degeneration, such as accumulation of vesicles in axons and focal myelin splitting and folding, was observed by transmission electron microscopy at onset; severe degeneration, such as axonal loss, myelin ovoid, and demyelination, increased at peak; and regenerative changes, such as remyelination and enlargement of Schwann cell cytoplasm, occurred at recovery. These results suggest that EAN rats have histopathological similarities to some types of GBS patients and that EAN rats are a useful model to understand the pathogenesis of GBS.

NRG1 type I dependent autoparacrine stimulation of Schwann cells in onion bulbs of peripheral neuropathies.

In contrast to acute peripheral nerve injury, the molecular response of Schwann cells in chronic neuropathies remains poorly understood. Onion bulb structures are a pathological hallmark of demyelinating neuropathies, but the nature of these formations is unknown. Here, we show that Schwann cells induce the expression of Neuregulin-1 type I (NRG1-I), a paracrine growth factor, in various chronic demyelinating diseases. Genetic disruption of Schwann cell-derived NRG1 signalling in a mouse model of Charcot-Marie-Tooth Disease 1A (CMT1A), suppresses hypermyelination and the formation of onion bulbs. Transgenic overexpression of NRG1-I in Schwann cells on a wildtype background is sufficient to mediate an interaction between Schwann cells via an ErbB2 receptor-MEK/ERK signaling axis, which causes onion bulb formations and results in a peripheral neuropathy reminiscent of CMT1A. We suggest that diseased Schwann cells mount a regeneration program that is beneficial in acute nerve injury, but that overstimulation of Schwann cells in chronic neuropathies is detrimental.

Intrathecal triamcinolone acetonide exerts anti-inflammatory effects on Lewis rat experimental autoimmune neuritis and direct anti-oxidative effects on Schwann cells.

BACKGROUND: Corticosteroids dominate in the treatment of chronic autoimmune neuropathies although long-term use is characterized by devastating side effects. METHODS: We introduce the intrathecal application of the synthetic steroid triamcinolone (TRIAM) as a novel therapeutic option in experimental autoimmune neuritis in Lewis rats RESULTS: After immunization with neuritogenic P2 peptide, we show a dose-dependent therapeutic effect of one intrathecal injection of 0.3 or 0.6 mg/kg TRIAM on clinical and electrophysiological parameters of neuritis with a lower degree of inflammatory infiltrates (T cells and macrophages) and demyelination in the sciatic nerve. In vitro studies in Schwann cell cultures showed an increased expression of IL-1 receptor antagonist and reduced expression of Toll-like receptor 4 after incubation with TRIAM as well as a protective effect of TRIAM against oxidative stress after H2O2 exposure. CONCLUSION: Intrathecal TRIAM application could be a novel immunomodulatory and potentially neuroprotective option for autoimmune neuropathies with a direct effect on Schwann cells.

FTY720 controls disease severity and attenuates sciatic nerve damage in chronic experimental autoimmune neuritis.

BACKGROUND: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an autoimmune-mediated inflammatory disease of the peripheral nervous system characterized by a response directed against certain myelin proteins and for which therapies are limited. Previous studies have suggested a beneficial role of FTY720, a sphingosine 1-phosphate (S1P) receptor agonist, known to deplete lymphocytes from the peripheral blood by sequestering them into lymph nodes, in the treatment of experimental autoimmune neuritis (EAN). Therefore, we investigated whether FTY720 is also beneficial in chronic experimental autoimmune neuritis (c-EAN), a recently developed rat model mimicking human CIDP. METHODS: c-EAN was induced in Lewis rats by immunization with S-palm P0(180-199) peptide. Rats were treated with FTY720 (1 mg/kg) or vehicle intraperitoneally once daily from the onset of clinical signs for 18 days; clinical signs were assessed daily until 60 days post-immunization (dpi). Electrophysiological and histological features were examined at different time points. We also evaluated the serum levels of different pro- and anti-inflammatory cytokines by ELISA or flow cytometry at 18, 40, and 60 dpi. RESULTS: Our data demonstrate that FTY720 decreased the severity and abolished the chronicity of the disease in c-EAN rats. Therapeutic FTY720 treatment reversed electrophysiological and histological anomalies, suggesting that myelinated fibers were subsequently preserved, it inhibited macrophage and IL-17+ cell infiltration in PNS, and it significantly reduced circulating pro-inflammatory cytokines. CONCLUSIONS: FTY720 treatment has beneficial effects on c-EAN, a new animal model mimicking human CIDP. We have shown that FTY720 is an effective immunomodulatory agent, improving the disease course of c-EAN, preserving the myelinated fibers, attenuating the axonal degeneration, and decreasing the number of infiltrated inflammatory cells in peripheral nerves. These data confirm the interest of testing FTY720 or molecules targeting S1P in human peripheral neuropathies.

The effects of vasoactive intestinal peptide in the rat model of experimental autoimmune neuritis and the implications for treatment of acute inflammatory demyelinating polyradiculoneuropathy or Guillain-Barré syndrome.

BACKGROUND: Guillain-Barré syndrome is an acute inflammatory demyelinating polyneuropathy that is characterized histologically by demyelination of peripheral nerves and nerve roots, infiltrates of T lymphocytes, and an inflammatory response that includes macrophage infiltrates. The aim of this study was to evaluate the effects of vasoactive intestinal peptide (VIP) in a rat model of experimental autoimmune neuritis (EAN). METHODS: Forty male Lewis rats were divided into a control group (N=10), an EAN group (N=10), an EAN group treated with 15 nmol of VIP (N=10), and an EAN group treated with 30 nmol of VIP (N=10). The rat model was created by subcutaneous injection of P2 polypeptide (200 µg P257-81) into the base of the tail. Intraperitoneal injection of VIP was given on day 7. Rats were weighed and functionally evaluated using an EAN score (0-10). On day 16, the rats were euthanized. The sciatic nerve was examined histologically and using immunohistochemistry with antibodies against CD8, CD68, and forkhead box p3 (Foxp3). Serum concentrations of IL-17 and interferon-α (IFN-α) were measured by ELISA on day 16 after creating the EAN model. RESULTS: The VIP-treated EAN groups had increased body weight and improved EAN scores compared with the untreated EAN group. CD8-positive and CD68-positive cells were significantly reduced in the EAN group treated with 30 nmol of VIP compared with 15 nmol of VIP. Foxp3-positive cells were significantly decreased in both EAN groups treated with VIP, and serum concentrations of IL-17 and IFN-α were significantly lower compared with the untreated EAN group (P<0.05). CONCLUSION: In a rat model of EAN, treatment with VIP resulted in functional improvement, reduced nerve inflammation, and decreased serum levels of inflammatory cytokines.

Trapped in the epineurium: early entry into the endoneurium is restricted to neuritogenic T cells in experimental autoimmune neuritis.

BACKGROUND: Autoimmune polyneuropathies are acquired inflammatory disorders of the peripheral nervous system (PNS) characterized by inflammation, demyelination, and axonal degeneration. Although the pathogenesis has not been fully elucidated, T cells recognizing self-antigens are believed to initiate inflammation in a subgroup of patients. However, the route and time of T cell entry into the PNS have not yet been described in detail. In this study, we analyzed both kinetics as well as localization of retrovirally transfected green fluorescent protein (GFP)-expressing neuritogenic T lymphocytes in experimental autoimmune neuritis (EAN). METHODS: T lymphocytes obtained from rats following EAN induction by immunization with peripheral nerve protein peptide P255-78 were retrovirally engineered to express GFP. Non-specific T cells were negatively selected by in vitro restimulation, whereas GFP-expressing neuritogenic T cells (reactive to P255-78) were adoptively transferred into healthy rats (AT-EAN). Antigen-specific T cell tracking and localization was performed by flow cytometry and immunohistochemistry during the course of disease. RESULTS: After induction of autoimmune neuritis, P2-reactive T cells were detectable in the liver, spleen, lymph nodes, lung, peripheral blood, and the sciatic nerves with distinct kinetics. A significant number of GFP+ T cells appeared early in the lung with a peak at day four. In the peripheral nerves within the first days, GFP-negative T cells rapidly accumulated and exceeded the number of GFP-expressing cells, but did not enter the endoneurium. Very early after adoptive transfer, T cells are found in proximity to peripheral nerves and in the epineurium. However, only GFP-expressing neuritogenic T cells are able to enter the endoneurium from day five after transfer. CONCLUSIONS: Our findings suggest that neuritogenic T cells invade the PNS early in the course of disease. However, neuritogenic T cells cross the blood-nerve barrier with a certain delay without preference to dorsal roots. Further understanding of the pathophysiological role of autoagressive T cells may help to improve therapeutic strategies in immune-mediated neuropathies.

Decitabine induces regulatory T cells, inhibits the production of IFN-gamma and IL-17 and exerts preventive and therapeutic efficacy in rodent experimental autoimmune neuritis.

Guillain-Barré syndrome (GBS) is an immune-mediated acute disorder of the peripheral nervous system. Despite treatment, there is an associated mortality and severe disability in 9 to 17% of the cases. Decitabine (DAC) is a hypomethylating drug used in myelodisplastic syndrome, that has been shown to exert immunomodulatory effects. We have evaluated the effects of DAC in two rodent models of GBS, the Experimental Allergic Neuritis (EAN). Both prophylactic and therapeutic treatment with DAC ameliorated the clinical course of EAN, increasing the numbers of thymic regulatory T cells and reducing the production of proinflammmatory cytokines. Our data suggest the possible use of decitabine for the treatment of GBS.

Therapeutic Effect of CD4+CD25+ Regulatory T Cells Amplified In Vitro on Experimental Autoimmune Neuritis in Rats.

BACKGROUND/AIMS: This study aimed to explore whether the adoptive transfusion of autologous CD4+CD25+ regulatory T cells (CD4+CD25+ Tregs) has a therapeutic effect on Experimental autoimmune neuritis (EAN) model rats, and it provides new experimental and theoretical bases for the immunotherapy of Guillain-Barre syndrome (GBS). METHODS: CD4+CD25+ Tregs were sorted from the spleens of rats using immunomagnetic bead separation techniques combined with flow cytometry. Their in vitro inhibitory function was determined using a lymphocyte proliferation inhibition test, and their purity was confirmed by flow cytometry. Cells were stimulated using CD3/CD28 monoclonal antibodies and were cultured in culture medium containing interleukin 2 (IL-2), transforming growth factor-ß (TGF-ß) and rapamycin. After 15 days of amplification, CD4+CD25+ Tregs were collected and transfused into EAN model rats. Changes in the pathology and electron microscopical morphology of rat sciatic nerves in the normal group, untreated group, low-dose group (2 × 107) and high-dose group (4 × 107) were observed, and the expression of CD4+CD25+FOXP3 in peripheral blood in the four groups of rats was detected by flow cytometry. RESULTS: Compared with rats in the untreated group, rats in the treatment groups had significantly reduced infiltration of inflammatory cells in the sciatic nerve, as well as myelin and axonal damage. Additionally, the CD4+CD25+ Tregs levels in peripheral blood were significantly higher than those in the untreated group (P< 0. 05). Moreover, the therapeutic effect became more significant with an increase in the dose of adoptive transfusion. CONCLUSION: Adoptive transfusion of CD4+CD25+ Tregs into EAN model rats has significant therapeutic effects.

Capsaicin-enriched diet ameliorates autoimmune neuritis in rats.

BACKGROUND: Autoimmune neuropathies are common PNS disorders and effective treatment is challenging. Environmental influence and dietary components are known to affect the course of autoimmune diseases. Capsaicin as pungent component of chili-peppers is common in human nutrition. An influence of capsaicin on autoimmune diseases has been postulated. METHODS: We tested capsaicin in the animal model of experimental autoimmune neuritis (EAN) in Lewis rat. Rats were immunized with P2-peptide and were treated with capsaicin in different preventive settings. Electrophysiological, histological, and molecular biological analyses of the sciatic nerve were performed to analyze T-cell and macrophage cell count, TRPV1, and cytokine expression. Moreover, FACS analyses including the intestinal immune system were executed. RESULTS: We observed an immunomodulatory effect of an early preventive diet-concept, where a physiological dosage of oral capsaicin was given 10 days before immunization in EAN. A reduced inflammation of the sciatic nerve was significant detectable clinically, electrophysiologically (CMAPs reduced in control group p < 0.01; increase of nerve conduction blocks in control group p < 0.05), histologically (significant reduction of T-cells, macrophages and demyelination), and at cytokine level. In contrast, this therapeutic effect was missing with capsaicin given from the day of immunization onwards. As possible underlying mechanism, we were able to show changes in the expression of the capsaicin receptor in the sciatic nerve and the small intestine, as well as altered immune cell populations in the small intestine. CONCLUSION: This is the first report about the immunomodulatory effect of the common nutrient, capsaicin, in an experimental model for autoimmune neuropathies.