Jun-Yang Wang

GABA, a natural immunomodulator of T lymphocytes

gamma-aminobutyric acid (GABA) is the main neuroinhibitory transmitter in the brain. Here we show that GABA in the extracellular space may affect the fate of pathogenic T lymphocytes entering the brain. We examined in encephalitogenic T cells if they expressed functional GABA channels that could be activated by the low (nM-1 microM), physiological concentrations of GABA present around neurons in the brain. The cells expressed the alpha1, alpha4, beta2, beta3, gamma1 and delta GABAA channel subunits and formed functional, extrasynaptic-like GABA channels that were activated by 1 microM GABA. 100 nM and higher GABA concentrations decreased T cell proliferation. The results are consistent with GABA being immunomodulatory.

FoxA1 directs the lineage and immunosuppressive properties of a novel regulatory T cell population in EAE and MS

The defective generation or function of regulatory T (Treg) cells in autoimmune disease contributes to chronic inflammation and tissue injury. We report the identification of FoxA1 as a transcription factor in T cells that, after ectopic expression, confers suppressive properties in a newly identified Treg cell population, herein called FoxA1+ Treg cells. FoxA1 bound to the Pdl1 promoter, inducing programmed cell death ligand 1 (Pd-l1) expression, which was essential for the FoxA1+ Treg cells to kill activated T cells. FoxA1+ Treg cells develop primarily in the central nervous system in response to autoimmune inflammation, have a distinct transcriptional profile and are CD4+FoxA1+CD47+CD69+PD-L1hiFoxP3−. Adoptive transfer of stable FoxA1+ Treg cells inhibited experimental autoimmune encephalomyelitis in a FoxA1–and Pd-l1–dependent manner. The development of FoxA1+ Treg cells is induced by interferon-β (IFN-β) and requires T cell–intrinsic IFN-α/β receptor (Ifnar) signaling, as the frequency of FoxA1+ Treg cells was reduced in Ifnb−/− and Ifnar−/− mice. In individuals with relapsing-remitting multiple sclerosis, clinical response to treatment with IFN-β was associated with an increased frequency of suppressive FoxA1+ Treg cells in the blood. These findings suggest that FoxA1 is a lineage-specification factor that is induced by IFN-β and supports the differentiation and suppressive function of FoxA1+ Treg cells.

Lack of Neuronal IFN-β-IFNAR Causes Lewy Body- and Parkinson’s Disease-like Dementia

Summary Neurodegenerative diseases have been linked to inflammation, but whether altered immunomodulation plays a causative role in neurodegeneration is not clear. We show that lack of cytokine interferon-β (IFN-β) signaling causes spontaneous neurodegeneration in the absence of neurodegenerative disease-causing mutant proteins. Mice lacking Ifnb function exhibited motor and cognitive learning impairments with accompanying α-synuclein-containing Lewy bodies in the brain, as well as a reduction in dopaminergic neurons and defective dopamine signaling in the nigrostriatal region. Lack of IFN-β signaling caused defects in neuronal autophagy prior to α-synucleinopathy, which was associated with accumulation of senescent mitochondria. Recombinant IFN-β promoted neurite growth and branching, autophagy flux, and α-synuclein degradation in neurons. In addition, lentiviral IFN-β overexpression prevented dopaminergic neuron loss in a familial Parkinson’s disease model. These results indicate a protective role for IFN-β in neuronal homeostasis and validate Ifnb mutant mice as a model for sporadic Lewy body and Parkinson’s disease dementia.

Tumor necrosis factor-α of Red nucleus involved in the development of neuropathic allodynia.

The pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with the generation of inflammatory and neuropathic pain. The current study aims to investigate the expression of TNF-α in the brain of rats with spared nerve injury (SNI), a neuropathic pain model with the lesion of common peroneal and tibial nerves. Two weeks following SNI, the immunohistochemical results identified that the expression level of TNF-α in the Red nucleus (RN) of SNI rats was apparently higher than that of sham-operated rats. To further study the roles of TNF-α in the development of neuropathic pain, different doses of anti-TNF-α antibody (20, 2.0 and 0.2 μg/ml) were microinjected into the RN contralateral to the nerve injury side of SNI rats. The results showed that the 50% paw withdrawal threshold (von Frey test) of SNI rats were increased by 20 and 2.0 μg/ml anti-TNF-α antibody as compared with that of the basic value and control groups (P<0.05), the analgesic effect lasted for 50 and 30 min, respectively. However, no significant analgesic effect was observed after 0.2 μg/ml antibody was microinjected into the RN. These results suggest that the TNF-α of RN is involved in the development of neuropathic allodynia in SNI rats.

Roles of different subtypes of opioid receptors in mediating the ventrolateral orbital cortex opioid-induced inhibition of mirror-neuropathic pain in the rat

Previous studies have demonstrated that opioid receptors in the prefrontal ventrolateral orbital cortex (VLO) are involved in anti-nociception. The aim of this current study was to examine whether opioid receptors in the VLO have effects on the hypersensitivity induced by contralateral L5 and L6 spinal nerve ligation (SNL), termed as mirror neuropathic pain (MNP) in the male rat. Morphine (1.0, 2.5, 5.0 microg) microinjected into the VLO contralateral to the SNL depressed the mechanical paw withdrawal assessed by von Frey filaments and the cold plate (4 degrees C)-induced paw lifting in a dose-dependent manner on the side without SNL. These effects were antagonized by microinjection of the non-selective opioid receptor antagonist naloxone (1.0 mug) into the same VLO site. Microinjection of endomorphin-1 (5.0 microg), a highly selective mu-opioid receptor agonist, and [d-Ala(2), d-Leu(5)]-enkephalin (DADLE, 10 microg), a delta-/mu-receptor agonist, also depressed the MNP. The effects of both drugs were blocked by selective mu-receptor antagonist beta-funaltrexamine (beta-FNA, 3.75 microg), but the effect of the DADLE was not influenced by the selective delta-receptor antagonist naltrindole (5.0 microg). Microinjection of the kappa-opioid receptor agonist spiradoline mesylate salt (U-62066) (100 microg) had no effect on the MNP. These results suggest that the VLO is involved in opioid-induced inhibition of the MNP and the effect is mediated by mu- (but not delta- and kappa-) opioid receptors.

μ- but not δ- and κ-opioid receptors in the ventrolateral orbital cortex mediate opioid-induced antiallodynia in a rat neuropathic pain model

Previous studies have indicated that the ventrolateral orbital cortex (VLO) is involved in opioid-mediated antinociception in the tail flick test and formalin test. The aim of the current study was to examine the effect of opioids microinjected into the VLO on allodynia in the rat L5/L6 spinal nerve ligation (SNL) model of neuropathic pain and determine the roles of different subtypes of opioid receptors in this effect. The allodynia was assessed by both mechanical (von Frey filaments) and cold plate (4 °C) stimuli. Morphine (1.0, 2.5, and 5.0 μg) microinjected into the VLO contralateral to the nerve ligation dose-dependently depressed the mechanical and cold allodynia and these effects were reversed by nonselective opioid receptor antagonist naloxone (1.0 μg) administrated into the same site. Microinjection of endomorphin-1 (5.0 μg), a highly selective μ-opioid receptor agonist, and [d-Ala2, d-Leu5]-enkephalin (DADLE, 10 μg), a δ-/μ-opioid receptor agonist, also depressed the allodynia, and the effects of both drugs were blocked by selective μ-receptor antagonist β-funaltrexamine (β-FNA, 3.75 μg), but the effects of DADLE were not influenced by the selective δ-receptor antagonist naltrindole (5.0 μg). Microinjection of U-62066 (100 μg), a κ-opioid receptor agonist, into the VLO had no effect on the allodynia. These results suggest that the VLO is involved in opioid-induced antiallodynia and μ- but not δ- and κ-opioid receptor mediates these effects in the rat with neuropathic pain.

NF-κB, ERK, p38 MAPK and JNK contribute to the initiation and/or maintenance of mechanical allodynia induced by tumor necrosis factor-alpha in the red nucleus

Previous studies have demonstrated that tumor necrosis factor-alpha (TNF-α) in the red nucleus (RN) plays facilitated roles in the development of abnormal pain. Here, the roles of nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in TNF-α-evoked mechanical allodynia were investigated. Repeated microinjection of recombinant rat TNF-α (20 ng daily for 3 days) into the unilateral RN of normal rats induced a significant mechanical allodynia in the contralateral but not ipsilateral hind paw at the fifth day and disappeared 24h later. Re-injection of a single bolus of 20 ng TNF-α into the same RN reproduced this mechanical allodynia within 30 min, which was used as a pain model for further experiments. Immunohistochemistry demonstrated that NF-κB, phospho-ERK (p-ERK) and p-p38 MAPK in the RN were significantly up-regulated at 1h after TNF-α microinjection, the up-regulations of NF-κB and p-ERK but not p-p38 MAPK remained at high levels till 4h later. A significant up-regulation of p-JNK occurred at 4h (but not 1h) after TNF-α microinjection, which was later than those of NF-κB, p-ERK and p-p38 MAPK. Pre-treatment with NF-κB inhibitor PDTC, ERK inhibitor PD98059 or p38 MAPK inhibitor SB203580 at 30 min before TNF-α microinjected into the RN completely prevented TNF-α-evoked mechanical allodynia. Pre-treatment with JNK inhibitor SP600125 did not prevent but reversed TNF-α-evoked mechanical allodynia during the subsequent detection time. Post-treatment with PDTC, PD98059 or SP600125 (but not SB203580) at 4h after TNF-α microinjected into the RN significantly reversed TNF-α-evoked mechanical allodynia. These results further prove that TNF-α in the RN plays a crucial role in the development of abnormal pain, and the algesic effect of TNF-α is initiated through activating NF-κB, ERK and p38 MAPK. The later maintenance of TNF-α-evoked mechanical allodynia mainly relies on the activation of NF-κB, ERK and JNK, but not p38 MAPK.

Transforming growth factor-beta in the red nucleus plays antinociceptive effect under physiological and pathological pain conditions.

Previous studies have demonstrated that the red nucleus (RN) participates in the modulation of neuropathic pain and plays both a facilitated role by pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β), and an inhibitory role through the anti-inflammatory cytokine IL-10. In this study, we sought to investigate the expressions and roles of transforming growth factor-beta (TGF-β), a potent anti-inflammatory cytokine, as well as its type 1 receptor (TGF-β-R1) in the RN in normal and neuropathic pain rats. Immunohistochemistry showed that TGF-β and TGF-β-R1 were constitutively expressed in the RN of normal rats, and co-localized with neurons and all three glial cell types, astrocytes, microglia and oligodendrocytes. Following spared nerve injury (SNI), the expression levels of TGF-β and TGF-β-R1 were significantly down-regulated in the RN contralateral (but not ipsilateral) to the nerve injury side of rats at one week and reached the lowest level at two weeks after SNI, and both of them were co-localized with neurons and oligodendrocytes but not with astrocytes and microglia. Microinjection of different doses of anti-TGF-β antibody (250, 125, 50 ng) into the unilateral RN of normal rats dose-dependently decreased the mechanical withdrawal threshold of contralateral (but not ipsilateral) hind paw and induced significant mechanical hypersensitivity, which was similar to mechanical allodynia induced by peripheral nerve injury. In contrast, microinjection of different doses of recombinant rat TGF-β1 (500, 250, 100 ng) into the RN contralateral to the nerve injury side of SNI rats dose-dependently increased the paw withdrawal threshold and significantly alleviated mechanical allodynia induced by SNI. These results suggest that TGF-β in the RN participates in nociceptive processing and plays antinociceptive effects under normal physiological condition and in the development of neuropathic pain induced by SNI.

Mu-Opioid Receptor in the Nucleus Submedius: Involvement in Opioid-Induced Inhibition of Mirror-Image Allodynia in a Rat Model of Neuropathic Pain

The current study investigated the roles of various subtypes of opioid receptors expressed in the thalamic nucleus submedius (Sm) in inhibition of mirror-image allodynia induced by L5/L6 spinal nerve ligation in rats. Morphine was microinjected into the Sm, which produced a dose-dependent inhibition of mirror-image allodynia; this effect was antagonized by pretreatment with non-selective opioid receptor antagonist naloxone. Microinjections of endomorphin-1 (μ-receptor agonist), or [d-Ala2, d-Leu5]-enkephalin (DADLE, δ-/μ-receptor agonist), also inhibited mirror-image allodynia, and these effects were blocked by the selective μ-receptor antagonist, β-funaltrexamine hydrochloride. The DADLE-induced inhibition, however, was not influenced by the δ-receptor antagonist naltrindole. The κ-receptor agonist, spiradoline mesylate salt, failed to alter the mirror-image allodynia. These results suggest that Sm opioid receptor signaling is involved in inhibition of mirror-image allodynia; this effect is mediated by μ- (but not δ- and κ-) opioid receptors in the rat model of neuropathic pain.

Thalamic nucleus submedius receives GABAergic projection from thalamic reticular nucleus in the rat.

GABAergic projection from thalamic reticular nucleus to thalamic nucleus submedius in the medial thalamus of the rat was studied by using immunohistochemistry for GABA, retrograde labeling with Fluoro-Gold combined with immunohistochemistry for GABA, and anterograde labeling with biotinylated dextranamine. Immunohistochemistry displayed that only GABA immunoreactive terminals were observed in the thalamic nucleus submedius, while GABA immunoreactive neuronal cell bodies were located in the thalamic reticular nucleus and lateral geniculate nucleus. Injection of Fluoro-Gold into the thalamic nucleus submedius resulted in massive retrogradely labeled neuronal cell bodies in the rostroventral portion of the ipsilateral thalamic reticular nucleus and a few in the contralateral thalamic reticular nucleus, and most of these cell bodies showed GABA immunopositive staining. Many biotinylated dextranamine anterogradely labeled fibers and terminals in the thalamic nucleus submedius were observed after injection of biotinylated dextranamine into the thalamic reticular nucleus. The present results provide a morphological evidence for a hypothesis that a disinhibitory effect on output neurons elicited by opioid or 5-hydroxytryptamine inhibiting a GABAergic terminal in the thalamic nucleus submedius may lead to activation of the descending inhibitory system and depression of the nociceptive inputs at the spinal cord level.