Bacillus-derived poly-γ-glutamic acid reciprocally regulates the differentiation of T helper 17 and regulatory T cells and attenuates experimental autoimmune encephalomyelitis.

Forkhead box protein 3 (FoxP3(+)) regulatory T (T(reg)) cells and interleukin (IL)-17-producing T helper 17 (Th17) cells have opposing effects on autoimmunity, as the former are crucial for maintaining self-tolerance while the latter play a key role in precipitating inflammatory autoimmune diseases. Here we report that Bacillus-derived poly-γ-glutamic acid (γ-PGA) signals naive CD4(+) T cells to promote the selective differentiation of T(reg) cells and to suppress the differentiation of Th17 cells. The γ-PGA inducibility of FoxP3 expression was due partially to transforming growth factor (TGF)-ß induction through a Toll-like receptor (TLR)-4/myeloid differentiating factor 88 (MyD88)-dependent pathway. However, this pathway was dispensable for γ-PGA suppression of Th17 differentiation. γ-PGA inhibited IL-6-driven induction of Th17-specific factors including signal transducer and activator of transcription-3 (STAT-3) and retinoic acid-related orphan receptor γt (RORγt) while up-regulating the STAT-3 inhibitor suppressor of cytokine signalling 3 (SOCS3). Importantly, in vivo administration of γ-PGA attenuated the symptoms of experimental autoimmune encephalomyelitis and at the same time reduced Th17 cell infiltrates in the central nervous system. Thus, we have identified the microbe-associated molecular pattern, γ-PGA, as a novel regulator of autoimmune responses, capable of promoting the differentiation of anti-inflammatory T(reg) cells and suppressing the differentiation of proinflammatory Th17 cells. These findings draw attention to the potential of γ-PGA for treating Th17 cell-mediated autoimmune diseases.

Bacillus-derived poly-γ-glutamic acid attenuates allergic airway inflammation through a Toll-like receptor-4-dependent pathway in a murine model of asthma.

BACKGROUND: Asthma is an inflammatory disease of the airways that is mediated by Th2 responses. Poly-γ-glutamic acid (γ-PGA) is an extracellular polymeric compound that is synthesized by Bacillus cells. Previously, we found that γ-PGA promoted Th1 cell development in a manner dependent on antigen-presenting cells, but inhibited Th2 cell development. OBJECTIVE: To investigate the effect of γ-PGA on dendritic cells (DCs), and its potential for treating Th2-mediated allergic asthma. METHODS: Wild-type, Toll-like receptor (TLR)-2 deficient, and TLR-4-defective mice were used. DCs derived from the bone marrow and extracted from the lung were stimulated with γ-PGA and assayed for the expression of signalling molecules, costimulatory molecules, and cytokines. Mice were sensitized and challenged with ovalbumin (OVA) to induce asthma. They were repeatedly injected intranasally with γ-PGA before and during the challenge period, and inflammation and structural remodelling of the airways were examined. RESULTS: γ-PGA selectively signalled conventional DCs to activate NF-κB and mitogen-activated protein kinase, leading to the up-regulation of CD86, CD40, and IL-12, but not IL-10 and IL-6. These effects of γ-PGA were dependent on TLR-4 and independent of TLR-2. Importantly, the intranasal administration of γ-PGA to OVA-sensitized/challenged mice reduced the airway hyperresponsiveness and allergic inflammation such as leucocyte influx, goblet cell hyperplasia, eosinophilia, and Th2 cytokine production. In addition to lowered IgE titres, the treatment of mice with γ-PGA significantly reduced the multiplication and Th2 polarization of mediastinal lymph node T cells upon allergen-specific restimulation. These anti-asthmatic effects of γ-PGA were also abolished in TLR-4-defective mice. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicate that γ-PGA activates DCs to favour Th1 cell induction through a TLR-4-dependent pathway and alleviates pathologic symptoms in a Th2-biased asthmatic model. These findings highlight the potential of γ-PGA for the treatment of asthma and other allergic disease in which Th2 polarization plays an important role.

Requirement of de novo protein synthesis for aminopterin-induced apoptosis in a mouse myeloma cell line.

Cells synthesize nucleotides through de novo and salvage pathways that require the activities of dihydrofolate reductase (DHFR) and hypoxanthine-guanine phosphoribosyltransfease (HGPRT), respectively. Aminopterin, an inhibitor of dihydrofolate reductase, has been demonstrated to allow HGPRT(-) cells to be negatively selected. However, the pathway by which aminopterin leads to cell death remains to be clarified. In this study, we characterized features of cellular responses induced by aminopterin treatment in P3-X63-Ag8.653, a mouse HGPRT(-) myeloma cell line. Upon treatment with aminopterin, the cells readily underwent an apoptotic process, as assessed by DNA fragmentation assay and electron microscopic analysis. Aminopterin-induced apoptosis was drastically reduced by addition of actinomycin D and cycloheximide, indicating that active RNA and protein synthesis is required for the apoptotic effect of aminopterin. Interestingly, the induction of c-myc gene expression preceded the activity of DNA fragmentation in aminopterin-treated cells. Taken together, these results suggest that cells deficient in the salvage pathway of purine biosynthesis are susceptible to aminopterin-induced apoptosis that requires de novo synthesis of proapoptotic factors, including Myc oncoprotein.

Modification of cardiovascular response of adenosine A1 receptor agonist by cyclic AMP in the spinal cord of the rats.

This study was performed to investigate the influence of the spinal adenosine A1 receptors on the central regulation of blood pressure (BP) and heart rate (HR), and to define whether its mechanism is mediated by cyclic AMP (cAMP) or cyclic GMP (cGMP). Intrathecal (i.t.) administration of drugs at the thoracic level were performed in anesthetized, artificially ventilated male Sprague-Dawley rats. Injection (i.t.) of adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA; 1, 5 and 10 nmol) produced dose dependent decrease of BP and HR. Pretreatment with a cAMP analogue, 8-bromo-cAMP, attenuated the depressor and bradycardiac effects of CHA (10 nmol), but not with cGMP analogue, 8-bromo-cGMP. These results suggest that adenosine A1 receptor in the spinal cord plays an inhibitory role in the central cardiovascular regulation and that this depressor and bradycardiac actions are mediated by cAMP.

Modification of cardiovascular response of adenosine A2 receptor agonist by adenylate cyclase in the spinal cord of rats.

This study was performed to investigate the influence of spinal adenosine A2 receptors on the central regulation of blood pressure (BP) and heart rate (HR), and to define whether its mechanism is mediated by adenylate cyclase or guanylate cyclase. Intrathecal (i.t.) administration of drugs at the thoracic level were performed in anesthetized, artificially ventilated male Sprague-Dawley rats. Injection (i.t.) of adenosine A2 receptor agonist, 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA; 1, 2 and 3 nmol) produced a dose dependent decrease of BP and HR. Pretreatment with adenylate cyclase inhibitor, MDL-12,330, attenuated the depressor and bradycardiac effects of CPCA (2 nmol), but not with guanylate cyclase inhibitor, LY-83,583. These results suggest that adenosine A2 receptor in the spinal cord plays an inhibitory role in the central cardiovascular regulation and that the depressor and bradycardiac actions are mediated by adenylate cyclase.

Mediation of the cardiovascular response of adenosine A1 receptor through a GABA(B) receptor in the spinal cord of the rat.

Cardiovascular inhibitory effects induced by intrathecal (i.t.) administration of adenosine A1 receptor agonist and its modulation by cyclic AMP was suggested by our previous report. In this experiment, we examined the mediation of cardiovascular effects of adenosine A1 receptor by gamma-aminobutyric acid receptors A and B [GABA(A) and GABA(B)] in the spinal cord. I.t. administration of 10 nmol of N6-cyclohexyladenosine (CHA), an adenosine A1 receptor agonist, and pretreatment with bicuculline (10 nmol, i.t), a GABA(A) receptor antagonist, and 5-aminovaleric acid (50 nmol, i.t.), a GABA(B) receptor antagonist, prior to injection of CHA were performed in anesthetized, artificially ventilated Sprague-Dawley rats. I.t. injection of 50 nmol of 5-aminovaleric acid significantly attenuated the inhibitory cardiovascular effects of CHA but 10 nmol of bicuculline did not alter CHA-induced cardiovascular actions. It is suggested that cardiovascular responses of adenosine A1 receptor is mediated by GABA(B) receptor in the spinal cord.

Branching patterns of the arterial branches supplying the middle vascular pedicle of the sternocleidomastoid muscle: a topographic anatomical study with surgical applications for the use of pedicles osteomuscular flaps.

When making a sternocleidomastoid (SCM) osteomuscular flap to include the clavicle and determining the rotation arc of the osteomuscular flap, it is very important to know the location and the origin of the superior thyroid artery and the distribution pattern of the SCM branch. Accordingly, in this study, the 50 SCM muscles and their arteries were dissected in 26 Korean cadavers, and the results were analyzed. The average distances from the origin of the superior thyroid artery to the clavicular and sternal heads of the SCM muscle were 87.6 mm (57.7-123.8 mm) and 131.2 mm (99.7-166.8 mm), respectively. The average distance from the origin of the superior thyroid artery to the SCM branch entering the SCM muscle was 30.1 mm (16.0-37.7 mm). After entering the SCM muscle, the SCM branches of the superior thyroid artery bifurcated into the clavicular and sternal branches at a point located an average of 58.8 mm (28.4-130.4 mm) above the clavicle. The distribution patterns of the superior thyroid artery were classified into six types based on the branching order and the dual supplies to the SCM muscle. Among them, type I in which the laryngeal branch first divided from the superior thyroid artery was the most common case (36%).

Modification of cardiovascular responses to spinal GABA(B) receptor stimulation by cAMP and by K(ATP) channel blockade in anaesthetized rats.

1. Intrathecal (i.t.) injection of baclofen (30, 60 and 100 nmol), a GABA(B) receptor agonist, produced a dose-dependent decrease in blood pressure (BP) and heart rate (HR). 2. Pretreatment with 5-aminovaleric acid (50 nmol), a GABA(B) receptor antagonist, blocked the depressor and bradycardic effects of baclofen (100 nmol). 3. Pretreatment with 8-bromo-cAMP (10 nmol), a cAMP analogue, attenuated the depressor and bradycardic effects of baclofen (100 nmol), but not with 8-bromo-cGMP (10 nmol), a cGMP analogue. 4. In addition, pretreatment with glipizide (20 nmol), an ATP-sensitive K+ channel (K(ATP)) blocker, attenuated the depressor and bradycardic effects of baclofen (100 nmol). These results suggest that GABA(B) receptors in the spinal cord have an inhibitory role in the central cardiovascular regulation and that these depressive and bradycardic actions are modified by cAMP and by K(ATP) channel blockade.

Modification of cardiovascular responses to adenosine A1 receptor stimulation in the posterior hypothalamus of anaesthetized rats by cAMP and by GABA(B) receptor blockade.

1 Injection of N(6)-cyclohexyladenosine (CHA; 1, 5 and 10 nmol), an adenosine A1 receptor agonist, into the posterior hypothalamus of rats produced a dose-dependent decrease in blood pressure (BP) and heart rate (HR). 2 Pretreatment with 8-cyclopentyl-1,3-dimethylxanthine (CPDX; 50 nmol), an adenosine A1 receptor antagonist, blocked the depressor and bradycardic effects of CHA (10 nmol). 3 Pretreatment with 8-bromo-cyclic adenosine monophosphate (AMP) (10 nmol), a cAMP analogue, attenuated the depressor and bradycardic effect of CHA (10 nmol); 8-bromo-cyclic guanosine monophosphate (GMP) (10 nmol), a cGMP analogue, did not modify those effects of CHA. 4 In addition, pretreatment with 5-aminovaleric acid (25 nmol), a gamma-aminobutyric acid (GABA)(B) receptor antagonist, attenuated the depressor and bradycardic effects of CHA (10 nmol). 5 These results suggest that adenosine A1 receptors in the posterior hypothalamus have an inhibitory role in the central cardiovascular regulation and that these vasodepressive and bradycardic actions are modified by raised cAMP and by GABA(B) receptor inhibition.

Metallothionein suppresses collagen-induced arthritis via induction of TGF-beta and down-regulation of proinflammatory mediators.

Metallothionein is a low molecular weight, cysteine-rich, stress response protein that can act as an antioxidant and as an immunosuppressive agent in instances of antigen-dependent adaptive immunity. In this context, we assessed the therapeutic potential and mechanisms of action of metallothionein in a collagen-induced arthritis model. Repeated administration of metallothionein-I + II during the course of disease dramatically reduced the incidence and severity of the disease. Joint tissues isolated from boostered paws of metallothionein-I + II-treated mice expressed significantly reduced levels of proinflammatory mediators, such as tumour necrosis factor (TNF)-alpha and cyclooxygenase-2, when compared with those of control-treated mice. Lymph node cells obtained from metallothionein-I + II -injected mice exhibited a significant decrease in the proliferative response and a remarkable increase in tumour growth factor (TGF)-beta production in response to type II collagen. Taken together, these results suggest that metallothionein-I + II promote the development of type II collagen-specific, TGF-beta-producing cells to antagonize the expansion of arthritogenic cells. This could lead to local suppression of inflammatory responses by inhibiting the expression of proinflammatory molecules. Thus, this study demonstrates the suppressive effects of metallothionein on collagen-induced arthritis, and indicates that there may be a potential therapeutic application for manipulation of metallothionein during the treatment of autoimmune disorders.