Zhi-Yuan Zhang

Compound A, a Plant Origin Ligand of Glucocorticoid Receptors, Increases Regulatory T Cells and M2 Macrophages to Attenuate Experimental Autoimmune Neuritis with Reduced Side Effects

Experimental autoimmune neuritis (EAN) is a helper T cell-mediated autoimmune demyelinating inflammatory disease of the peripheral nervous system and serves as the animal model for human inflammatory demyelinating polyneuropathies. Compound A, a plant-derived phenyl aziridine precursor, was reported to activate glucocorticoid receptors to exert transrepression but not transactivation properties. In this study, we investigated the effects of Compound A in EAN rats. Compound A greatly suppressed paraparesis in EAN, even when administrated after the appearance of the first neurological signs. Accumulation of macrophages and lymphocytes, demyelination, and mRNA levels of inflammatory molecules in sciatic nerves of EAN were greatly attenuated by Compound A. In addition, Compound A inhibited progression of neuropathic pain and repressed microglia but not astrocyte activation and IL-1β and TNF-α up-regulation in EAN spinal cords. In EAN sciatic nerves, Compound A treatment increased numbers of anti-inflammatory M2 macrophages. Furthermore, Compound A induced the switch of macrophages from inflammatory M1 type to anti-inflammatory M2 type in vitro. In lymph nodes of EAN rats, Compound A depressed Th1 and Th17 cytokines, but increased Th2 cytokine and Foxp3 expression. An increase of Foxp3+/CD4+ regulatory T cells was seen in peripheral blood of EAN rats following Compound A treatment. In addition, Compound A did not cause a hyperglycemia effect in EAN rats as compared with the immunosuppressive steroid prednisolone. Therefore, our data demonstrated that Compound A could effectively suppress EAN with reduced side effects by attenuating inflammation, suggesting that Compound A could be a potent candidate for treatment of autoimmune neuropathies.

Parenchymal accumulation of CD163+ macrophages/microglia in multiple sclerosis brains

Reactive macrophages/microglia exert both protective or damaging effects in multiple sclerosis (MS), which contribute to the relapsing-remitting nature of MS. CD163 is considered a marker of M2 (alternatively activated) macrophages. In the MS brain, CD163(+) perivascular macrophages express molecules for antigen recognition and presentation. Here we further investigated the accumulation of CD163(+) macrophages/microglia in the parenchyma of MS brains. CD163 expression pattern was investigated in different lesions of brain tissue specimens from five MS brains and five neuropathologically unaffected controls by immunohistochemistry. In the parenchyma of normal brain samples, immunoreactivity (IR) of CD163 was absent. In acute active lesions and at the rim of chronic active lesions of MS, strong accumulation of CD163(+) macrophages/microglia was seen. In chronic inactive lesions and in the center of chronic active lesion, CD163(+) macrophages/microglia were rare. Further, double-labeling showed that parenchymal and perivascular CD163(+) macrophages/microglia were myelin basic protein positive and HLA-DR(+), suggesting that CD163(+) macrophages/microglia could ingest and present antigen. In addition, in vitro incubating macrophage RAW264.7 cells with myelin turned LPS-induced inflammatory macrophages into an anti-inflammatory phenotype, indicating that myelin basic protein positive, CD163(+) macrophages/microglia in MS might have anti-inflammatory effects. The parenchymal CD163(+) macrophages/microglia, which had the capacity for antigen ingestion and presentation, might contribute to the resolution of inflammation in MS.

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.

Oral Administration of Histone Deacetylase Inhibitor MS-275 Ameliorates Neuroinflammation and Cerebral Amyloidosis and Improves Behavior in a Mouse Model

Alzheimer disease is the most common neurodegenerative disease and the major cause of dementia. In addition to β-amyloid aggregation and hyperphosphorylated tau, neuroinflammation also plays important roles in the pathophysiology of this multifactorial disorder. Histone deacetylase catalyzes deacetylation of histones and has important roles in the regulation of gene expression. Histone deacetylase inhibitors have been reported to exhibit neuroprotective and anti-neuroinflammatory activities and have therapeutic effects in several animal models of neurodegenerative diseases. Here, an efficient benzamide histone deacetylase inhibitor, MS-275, was orally administered by gavage to transgenic APP/PS1 mice, an animal model of cerebral amyloidosis for Alzheimer disease. After 10 days of treatment, MS-275 significantly ameliorated microglial activation and β-amyloid deposition in cerebral cortex and/or hippocampus. This was associated with improved nesting behavior, an important affiliative/social behavior. MS-275 also attenuated inflammatory activation of a mouse macrophage cell line in vitro. These results suggest that MS-275 may be a therapeutic option for Alzheimer disease and other neuroinflammatory diseases.

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.

Oxidative Burst of Circulating Neutrophils Following Traumatic Brain Injury in Human

Besides secondary injury at the lesional site, Traumatic brain injury (TBI) can cause a systemic inflammatory response, which may cause damage to initially unaffected organs and potentially further exacerbate the original injury. Here we investigated plasma levels of important inflammatory mediators, oxidative activity of circulating leukocytes, particularly focusing on neutrophils, from TBI subjects and control subjects with general trauma from 6 hours to 2 weeks following injury, comparing with values from uninjured subjects. We observed increased plasma level of inflammatory cytokines/molecules TNF-α, IL-6 and CRP, dramatically increased circulating leukocyte counts and elevated expression of TNF-α and iNOS in circulating leukocytes from TBI patients, which suggests a systemic inflammatory response following TBI. Our data further showed increased free radical production in leukocyte homogenates and elevated expression of key oxidative enzymes iNOS, COX-2 and NADPH oxidase (gp91phox) in circulating leukocytes, indicating an intense induction of oxidative burst following TBI, which is significantly greater than that in control subjects with general trauma. Furthermore, flow cytometry assay proved neutrophils as the largest population in circulation after TBI and showed significantly up-regulated oxidative activity and suppressed phagocytosis rate for circulating neutrophils following brain trauma. It suggests that the highly activated neutrophils might play an important role in the secondary damage, even outside the injured brain. Taken together, the potent systemic inflammatory response induced by TBI, especially the intensively increase oxidative activity of circulating leukocytes, mainly neutrophils, may lead to a systemic damage, dysfunction/damage of bystander tissues/organs and even further exacerbate secondary local damage. Controlling these pathophysiological processes may be a promising therapeutic strategy and will protect unaffected organs and the injured brain from the secondary damage.

Valproic acid ameliorates inflammation in experimental autoimmune encephalomyelitis rats

Valproic acid (VPA) is a short-chain branched fatty acid with anti-inflammatory, neuro-protective and axon remodeling effects. Here we have studied effects of VPA in gpMBP(68-84)-induced experimental autoimmune encephalomyelitis (EAE). Both preventive (from Day 0 to Day 18) and therapeutic (from Day 7 to Day 18 or from Day 9 to Day 19) VPA (500 mg/kg, intra-gastric) administration to EAE rats once daily greatly reduced the severity and duration of EAE, and suppressed mRNA levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-17, matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS) and transcription factor T-bet, but increased levels of IL-4 mRNA in EAE spinal cords. Furthermore, preventive VPA treatment greatly attenuated accumulation of macrophages and lymphocytes in EAE spinal cords. VPA treatment altered the cytokine milieu of lymph nodes, modulating the Th profile from Th1 and Th17 to a profile of Th2 and regulatory T cells. In addition, in vitro study showed that VPA inhibited non-specific lymphocyte proliferation in a dose-dependent manner. In summary, our data demonstrated that VPA could suppress systemic and local inflammation to improve outcome of EAE, suggesting that VPA might be a candidate for treatment of multiple sclerosis.

MS-275, an histone deacetylase inhibitor, reduces the inflammatory reaction in rat experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune inflammatory demyelinating disease of the peripheral nervous system and serves as the animal model of human inflammatory demyelinating polyradiculoneuropathies. MS-275, a potent histone deacetylase inhibitor currently undergoing clinical investigations for various malignancies, has been reported to demonstrate promising anti-inflammatory activities. In our present study, MS-275 administration (3.5 mg/kg i.p.) to EAN rats once daily from the appearance of first neurological signs greatly reduced the severity and duration of EAN and attenuated local accumulation of macrophages, T cells and B cells, and demyelination of sciatic nerves. Further, significant reduction of mRNA levels of pro-inflammatory interleukin-1beta, interferon-gamma, interleukine-17, inducible nitric oxide synthase and matrix metalloproteinase-9 was observed in sciatic nerves of MS-275 treated EAN rats. In lymph nodes, MS-275 depressed pro-inflammatory cytokines as well, but increased expression of anti-inflammatory cytokine interleukine-10 and of foxhead box protein3 (Foxp3), a unique transcription factor of regulatory T cells. In addition, MS-275 treatment increased proportion of infiltrated Foxp3(+) cells and anti-inflammatory M2 macrophages in sciatic nerves of EAN rats. In summary, our data demonstrated that MS-275 could effectively suppress inflammation in EAN, through suppressing inflammatory T cells, macrophages and cytokines, and inducing anti-inflammatory immune cells and molecules, suggesting MS-275 as a potent candidate for treatment of autoimmune neuropathies.

FTY720 attenuates lesional interleukin-17+ cell accumulation in rat experimental autoimmune neuritis

Aims: Experimental autoimmune neuritis (EAN) is a well‐known animal model of human demyelinating polyneuropathies. Here we have studied the spatiotemporal accumulation of interleukin (IL)‐17+ cells in sciatic nerves of EAN rats and effects of FTY720, an agonist of sphingosine‐1‐phosphate (S1P) receptors. Methods: In this study, we examined the spatiotemporal expression of IL‐17 using immunohistochemistry and RT‐PCR, and analysed the IL‐17+ cell proportion in blood and lymph nodes using flow cytometry. Results: In sciatic nerves of EAN rats, IL‐17+ cells were mainly found to concentrate around blood vessels and IL‐17+ cell accumulation was temporally correlated with severity of neurological signs. FTY720, which has been shown to reduce severity of EAN, attenuated accumulation of IL‐17+ cells in sciatic nerves, decreased IL‐17+ cell proportion in peripheral blood, but increased IL‐17+ cell proportion in lymph nodes, suggesting the involvement of S1P signal pathway in regulating IL‐17+ cell trafficking. Conclusions: our data are consistent with the possibility that IL‐17+ cells might contribute to the pathogenesis of EAN and the S1P signal pathway may be involved in the in vivo trafficking of IL‐17+ cells.

Lesional Accumulation of CD163+ Macrophages/microglia in Rat Traumatic Brain Injury

A robust neuroinflammation, contributing to the development of secondary injury, is a common histopathological feature of traumatic brain injury (TBI). Characterization of leukocytic subpopulations contributing to the early infiltration of the damaged tissue might aid in further understanding of lesion development. Reactive macrophages/microglia can exert protective or damaging effects in TBI. CD163 is considered a marker of M2 (alternatively activated) macrophages. Therefore we investigated the accumulation of CD163(+) macrophages/microglia in the brain of TBI rats. TBI was induced in rats using an open skull weight-drop contusion model and the accumulation of CD163(+) cells was analyzed by immunohistochemistry. In normal rat brains, CD163 was expressed by meningeal, choroid plexus and perivascular macrophages. Significant parenchymal CD163(+) cell accumulation was observed two days post TBI and continuously increased in the investigated survival time. The accumulated CD163(+) cells were mainly distributed to the lesional areas and exhibited macrophage phenotypes with amoeboid morphologic characteristics but not activated microglial phenotypes with hypertrophic morphology and thick processes. Double-labeling experiments showed that most CD163(+) cells co-expressed heme oxygenase-1 (HO-1). In addition, in vitro incubating of macrophage RAW264.7 cells or primary peritoneal macrophages with hemoglobin- haptoglobin (Hb-Hp) complex suppressed LPS-induced inflammatory macrophages phenotype and induced CD163 and HO-1 upregulation, indicating that CD163(+) macrophages/microglia in TBI might have anti-inflammatory effects. And further study is necessary to identify functions of these cells in TBI.