Uwe Fauser

FTY720 ameliorates experimental autoimmune neuritis by inhibition of lymphocyte and monocyte infiltration into peripheral nerves

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune demyelinating inflammatory disease of the peripheral nervous system (PNS). T cells and macrophages are essential for the initiation and development of EAN. FTY720 acts as an agonist of sphingosine-1-phosphate receptors, resulting in inhibition of lymphocyte egress from secondary lymphoid tissues and thymocytes from thymus. This investigation describes the immunosuppressive effects of FTY720 in EAN, the animal model of autoimmune neuropathies. FTY720 (1 mg/kg body weight) completely suppressed paraparesis if administrated from the day of immunization. Furthermore, FTY720 greatly reduced the severity and duration of EAN even when administrated after the appearance of the first neurological sign. T cell, B cell, and macrophage infiltration and demyelination of sciatic nerves were significantly decreased in FTY720-treated EAN. Therefore, FTY720 might be a potential candidate for treatment of inflammatory neuropathies.

Mechanical allodynia and spinal up-regulation of P2X4 receptor in experimental autoimmune neuritis rats

Experimental autoimmune neuritis (EAN) is the animal model of acute inflammatory demyelinating polyradiculoneuropathy (AIDP) that is the most common subtype of Guillain-Barre syndrome (GBS). While neuropathic pain is a common symptom of GBS, its underlying mechanisms remain elusive. Central sensitization, particularly spinal glia (microglia and astrocytes) activation, is important for the initiation and maintenance of neuropathic pain. P2X(4) receptor (P2X(4)R) is an ATP-gated ion channel and its spinal up-regulation has been found to be crucial for the development of neuropathic pain following peripheral nerve injury. The initiation of mechanical allodynia in rat EAN was observed at day 9 before the onset of neurological signs. Maximal level of mechanical allodynia was observed from days 17-19 and then a slow recovery, long after the cessation of typical neurological signs of EAN, until day 37 was observed. Expression of P2X(4)R in lumbar spinal cords was studied by immunohistochemistry. P2X(4)R immunoreactivity (IR) was mainly seen in gray matter, particularly in the dorsal horn. Accumulation of P2X(4)R(+) cells in the lumbar dorsal horn was observed at day 9, reached the maximal level at day 17 and remained elevated until day 37 after immunization. Furthermore, a negative correlation between the density of P2X(4)R(+) cells in the lumbar dorsal horn with mean hind-paw withdrawal threshold in EAN rats was seen, indicating that P2X(4)R might contribute to EAN mechanical allodynia. Double staining revealed that almost all P2X(4)R(+) cells co-expressed CD68, a marker for reactive microglia, but not the astrocyte marker, glial fibrillary acidic protein (GFAP). Our data demonstrate that EAN induces mechanical allodynia and P2X(4)R expression in spinal microglia, suggesting that EAN is a good animal model for neuropathic pain in polyneuropathy and spinal microglia activation might participate in EAN-induced neuropathic pain.

Global hypomethylation defines a sub-population of reactive microglia/macrophages in experimental traumatic brain injury

Global alterations in gene expression have been observed in different traumatic brain injury (TBI) models and are considered of crucial importance to the development of subsequent tissue injury and repair. Cytosine methylation is a well-known process of endogenous DNA modification in mammals and the primary mechanism responsible for changes in epigenetic gene expression. Here we have investigated the early global spatio-temporal changes of the status of cellular DNA methylation in a rat TBI model by immunohistochemistry and analyzed the effects of dexamethasone on these changes. Global cellular hypomethylation was seen as early as day 1 in pannecrosis and day 2 in peripannecrosis following TBI. A sub-population of reactive microglia/macrophages was identified as the major source of hypomethylated cells by double-staining experiments. Further, peripheral administration of dexamethasone suppressed this lesional hypomethylation at day 2 post-injury. In sum, our data suggest that lesional hypomethylation defines a sub-population of activated microglia/macrophages involved in the early processes following traumatic brain injury.

Distribution of Foxp3(+) T-regulatory cells in experimental autoimmune neuritis rats.

T-regulatory cells expressing the forkhead box transcription factor 3 (Foxp3) play essential roles in immune homeostasis. Experimental autoimmune neuritis (EAN) is an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system. We investigated the distribution of Foxp3(+) cells in sciatic nerves, spleen and lymph nodes of EAN rats, and the influence of FTY720 on the localization of Foxp3(+) cells in EAN rats. In sciatic nerves of EAN rats, accumulation of Foxp3(+) cells was not seen during the pre-symptomatic phase (until Day 9) or during early or peak disease activity. In contrast, Foxp3(+) cell accumulation was regularly seen in the recovery from neurologic disease, suggesting a contribution of Foxp3(+) cells to the resolution of EAN. FTY720 was given at onset of EAN (Day 10) until Day 18. Following FTY720 administration, percentages of Foxp3(+) cells in EAN rats were increased in circulating blood, but reduced in lymph nodes compared to the PBS control. FTY720 treatment suppressed total numbers but increased percentages of Foxp3(+) cells in sciatic nerves of EAN rats. These data not only suggests a protective role of Foxp3(+) cells in EAN but also provides a potential way to alter localization of Foxp3(+) cells in vivo.

FTY720 attenuates accumulation of EMAP-II+ and MHC-II+ monocytes in early lesions of rat traumatic brain injury

FTY720 (Fingolimod) is a novel type of immunosuppressive agent inhibiting lymphocyte egress from secondary lymphoid tissues thereby causing peripheral lymphopenia. FTY720 can inhibit macrophage infiltration into inflammatory lesions under pathological conditions. FTY720 has been clinically evaluated for prophylaxis of allograft rejection and treatment of multiple sclerosis, showing promising immunosuppressive effects. A robust inflammatory response after traumatic brain injury (TBI) plays an important role in the secondary or delayed injuries of TBI. Here we have investigated by immunohistochemistry in a rat TBI model the effects of FTY720 on early cell accumulation into the inflammatory tissue response and on expression of major histo‐compatibility complex class II (MHC‐II) and endothelial‐monocyte activating polypeptide II (EMAP‐II). Accumulation of MHC‐II+ or EMAP‐II+ cells became significant 1 day after injury and continuously increased during the early time periods. Further, double‐staining experiments confirmed that the major cellular sources of MHC‐II were reactive macrophages, however MHC‐II+ cells only constituted a small subpopulation of reactive macrophages. Immediately after TBI, peripheral administration of FTY720 (1 mg/kg in 1 mL saline, every second day) significantly attenuated the accumulation of MHC‐II+ macrophages from Day 1 to 4 and significantly attenuated the accumulation of EMAP‐II+ macrophages/microglia at Day 4. Our findings show that FTY720 attenuates early accumulation of EMAP‐II+ and MHC‐II+ reactive monocytes following TBI, indicating that FTY720 might be a drug candidate to inhibit brain inflammatory reaction following TBI.

Improved outcome of EAN, an animal model of GBS, through amelioration of peripheral and central inflammation by minocycline

Experimental autoimmune neuritis (EAN) is a widely used animal model of the human acute inflammatory demyelinating polyradiculoneuropathy, which is the most common subtype of Guillain‐Barré Syndrome. EAN is pathologically characterized by breakdown of the blood‐nerve barrier, infiltration of reactive immune cells, local inflammation, demyelination in the peripheral nervous system and mechanical allodynia. Minocycline is known to have neuroprotective and anti‐inflammatory effects. Furthermore, relieve of neuropathic pain following minocycline administration was observed in a variety of animal models. Here, we investigated the effects of minocycline on rat EAN. Suppressive treatment with minocycline (50 mg/kg body weight daily immediately after immunization) significantly attenuated the severity and duration of EAN. Macrophage and T‐cell infiltration and demyelination in sciatic nerves of EAN rats treated with minocycline were significantly reduced compared to phosphate‐buffered saline (PBS)‐treated EAN rats. mRNA expressions of matrix metallopeptidase‐9, inducible nitric oxide synthase and pro‐inflammatory cytokines interleukin‐1 β and tumour necrosis factor‐α in EAN sciatic nerves were greatly decreased by administration of minocycline as well. Furthermore, minocycline attenuated mechanical allodynia in EAN rats and greatly suppressed spinal microglial activation. All together, our data showed that minocycline could effectively suppress the peripheral and spinal inflammation (immune activation) to improve outcome in EAN rats, which suggests that minocycline may be considered as a potential candidate of pharmacological treatment for autoimmune‐mediated neuropathies.

Early attenuation of lesional interleukin‐16 up‐regulation by dexamethasone and FTY720 in experimental traumatic brain injury

Aims: Interleukin‐16 (IL16) is an immunomodulatory cytokine, which induces lymphocyte migration, expression of proinflammatory IL1β, IL6 and tumour necrosis factor‐α, and modulates apoptosis. IL16 expression has been observed in several central nervous system diseases and may play a role in promoting inflammatory responses. Inflammation contributes considerably to secondary injury following traumatic brain injury (TBI). The aim of this study was to investigate early IL16 expression following experimental TBI and the effects of dexamethasone and FTY720 on early expression of IL16 in TBI rats. Methods: Rat TBI was induced using an open‐skull weight‐drop model. IL16 expression was studied by immunohistochemistry. TBI rats received an intraperitoneal injection of dexamethasone (1 mg/kg in 1 ml saline), FTY720 (1 mg/kg in 1 ml saline) or saline (1 ml) on Day 0 and Day 2 immediately after surgery. Results: Significant up‐regulation of IL16 was seen as early as 24 h post TBI. Double‐staining experiments, together with morphological classification, revealed a multicellular origin of IL16, including activated microglia/macrophages (about 85%), astrocytes (about 8%), neurones (about 5%) and granulocytes. Following peripheral administration of dexamethasone and FTY720, attenuated numbers of IL16+ cells were observed on Days 1 and 2 but not on Day 4 post TBI for dexamethasone and on Day 4 but not earlier for FTY720 respectively. Conclusions: Our observations reveal that dexamethasone and FTY720 have different but complementary effects on reduction of early IL16 expression following TBI.

Expression of Interleukin‐16 in Sciatic Nerves, Spinal Roots and Spinal Cords of Experimental Autoimmune Neuritis Rats

Experimental autoimmune neuritis (EAN) is a well‐known animal model of Guillain‐Barré Syndrome. In this study, we studied the spatiotemporal expression of interleukin‐16 (IL‐16) in the nervous system of EAN rats and pharmacological effects of minocycline on IL‐16 expressions in EAN rats. In sciatic nerves and dorsal/ventral roots of EAN rats, IL‐16+ cells, identified as macrophages and T cells, were mainly found to concentrate around blood vessels. However, in spinal cords, IL‐16+ microglial cells were mainly found in lumbar dorsal horns. Massive IL‐16+ cell accumulation in sciatic nerves and spinal roots was temporally correlated with severity of neurological signs of EAN. Furthermore, a strong correlation of IL‐16+ cell accumulation with local demyelination in perivascular areas of sciatic nerves, and significant reduction of IL‐16+ cell numbers in sciatic nerves and spinal cords by minocycline suggested a pathological contribution of IL‐16+ cells in EAN. Taken together, robust IL‐16+ cell accumulation in the nervous system and its temporal correlation with severity of neurological signs in EAN might suggest a pathological role of IL‐16 in EAN, which makes IL‐16 a potential pharmacological target.

Dexamethasone transiently attenuates up-regulation of endostatin/collagen XVIII following traumatic brain injury.

Endostatin/collagen XVIII is a specific inhibitor of endothelial proliferation and migration in vitro. It has also been shown to have anti-angiogenic activity and tumor growth inhibitory activity in vivo and in vitro. Here we studied expression of endostatin/collagen XVIII in a rat traumatic brain injury (TBI) model, focusing on the early phase. A significant up-regulation of endostatin/collagen XVIII in TBI began as early as 24 h post-TBI. Double-staining experiment revealed that the major resource of endostatin/collagen XVIII(+) cells in our TBI rat model was a subpopulation of reactivated microglia/macrophages. Our data further showed that dexamethasone attenuated up-regulation of endostatin/collagen XVIII expression at days 1 and 2, but not at day 4, post-TBI, indicating that dexamethasone might possess an early and transient influence to the angiogenesis following TBI.

Expression of RhoA by inflammatory macrophages and T cells in rat experimental autoimmune neuritis

RhoA is one of the best‐studied members of Rho GTPases. Experimental autoimmune neuritis (EAN), which is characterized by infiltration of T cells and macrophages into the peripheral nervous system, is an autoantigen‐specific T‐cell‐mediated animal model of human Guillain‐BarrŽ Syndrome. In this study, RhoA expression has been investigated in the dorsal/ventral roots of EAN rats by immunohistochemistry. A significant accumulation of RhoA+ cells was observed on Day 12, with a maximum around Day 15, correlating to the clinical severity of EAN. In dorsal/ventral roots of EAN, RhoA+ cells were seen in perivascular areas but also in the parenchyma. Furthermore, double‐labelling experiments showed that the major cellular sources of RhoA were reactive macrophages and T cells. In conclusion, this is the first demonstration of the presence of RhoA in the dorsal/ventral roots of EAN. The time courses and cellular sources of RhoA together with the functions of RhoA indicate that RhoA may function to facilitate macrophage and T‐cell infiltration in EAN and therefore could be a potential therapeutic target.