Lipoic acid modulates inflammatory responses of monocytes and monocyte-derived macrophages from healthy and relapsing-remitting multiple sclerosis patients.

Multiple sclerosis (MS) is a disabling neuroinflammatory disease. Its etiology is unknown, but both oxidative stress and inflammation appear to be involved in disease pathology. Macrophages are the predominant cell type in acute inflammatory brain lesions in MS. Macrophages produce proinflammatory and toxic molecules that promote demyelination and are key players in phagocytosis/degradation of myelin sheathes. Lipoic acid (LA) is an inexpensive, endogenously produced small molecule that exhibits antioxidant and anti-inflammatory effects. Treatment with LA is protective in MS and other inflammatory diseases. To examine the mechanism(s) by which LA may attenuate inflammatory lesion activity in MS, we used healthy control and MS cells to evaluate the effects of LA on levels of inflammatory cytokines, phagocytosis and the immunomodulator cyclic adenosine monophosphate (cAMP) in monocytes and monocyte-derived macrophages (MDMs). LA treatment resulted in a generally less inflammatory phenotype of monocytes and MDMs from healthy controls, and (to a lesser degree) MS donors. LA inhibited monocyte secretion of cytokines relevant to MS in monocytes, including tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß; LA effects on secretion of these cytokines in MDMs were mixed with inhibition of TNF-α and IL-6, but stimulation of IL-1ß, the latter perhaps as a result of altered macrophage polarization. LA inhibited phagocytosis in both monocytes and MDMs, and increased cAMP levels in monocytes. LA may modulate inflammatory cytokine secretion and phagocytosis via a cAMP-mediated mechanism.

Effects of lipoic acid on migration of human B cells and monocyte-enriched peripheral blood mononuclear cells in relapsing remitting multiple sclerosis.

Multiple sclerosis (MS) is a disease of the central nervous system characterized by inflammation and demyelination resulting in clinical disability. The rodent MS model suggests that infiltration of monocytes and B cells contributes to disease pathogenesis. Here, we compared the migratory capacity of human monocytes and B cells from healthy control (HC) and relapsing-remitting MS (RRMS) subjects, with or without lipoic acid (LA) treatment. Basal migration of monocyte-enriched PBMCs from RRMS subjects is significantly higher than HC PBMCs. LA treatment significantly inhibits monocyte and B cell migration in both cohorts, and may thus be therapeutically effective for treatment of MS.

Lipoic Acid Stimulates cAMP Production in Healthy Control and Secondary Progressive MS Subjects.

Lipoic acid (LA) exhibits antioxidant and anti-inflammatory properties; supplementation reduces disease severity and T lymphocyte migration into the central nervous system in a murine model of multiple sclerosis (MS), and administration in secondary progressive MS (SPMS) subjects reduces brain atrophy compared to placebo. The mechanism of action (MOA) of LA's efficacy in suppression of MS pathology is incompletely understood. LA stimulates production of the immunomodulator cyclic AMP (cAMP) in vitro. To determine whether cAMP could be involved in the MOA of LA in vivo, we performed a clinical trial to examine whether LA stimulates cAMP production in healthy control and MS subjects, and whether there are differences in the bioavailability of LA between groups. We administered 1200 mg of oral LA to healthy control, relapsing remitting MS (RRMS) and SPMS subjects, and measured plasma LA and cAMP levels in peripheral blood mononuclear cells (PBMCs). There were no significant differences between the groups in pharmacokinetic (PK) parameters. Healthy and SPMS subjects had increased cAMP at 2 and 4 h post-LA treatment compared to baseline, while RRMS subjects showed decreases in cAMP. Additionally, plasma concentrations of prostaglandin E2 (PGE2, a known cAMP stimulator) were significantly lower in female RRMS subjects compared to female HC and SPMS subjects 4 h after LA ingestion. These data indicate that cAMP could be part of the MOA of LA in SPMS, and that there is a divergent response to LA in RRMS subjects that may have implications in the efficacy of immunomodulatory drugs. This clinical trial, "Defining the Anti-inflammatory Role of Lipoic Acid in Multiple Sclerosis," NCT00997438, is registered at https://clinicaltrials.gov/ct2/show/record/NCT00997438 .

Donor variability may mask dimethyl fumarate's effects on nuclear factor E2-related factor 2 in human peripheral blood mononuclear cells.

OBJECTIVE: Dimethyl fumarate (DMF) is an anti-inflammatory and antioxidant drug used to treat multiple sclerosis, but its mechanism(s) of action are not fully understood. In central nervous system (CNS) cells, DMF activates nuclear factor E2-related factor 2 (Nrf2), perhaps ameliorating oxidative stress-induced damage. However, it is not known whether DMF also activates Nrf2 in peripheral immune cells, which are known to participate in CNS demyelination. We conducted a single observation study to determine whether DMF can activate Nrf2 in peripheral immune cells in vitro. RESULTS: We performed enzyme-linked immunosorbent assays to measure Nrf2 activation in nuclear extracts of human peripheral blood mononuclear cells treated with DMF at time points from 0 to 6 h, initially determining that DMF did not activate Nrf2, and that the mechanism(s) of action of DMF may thus differ in the periphery compared to the CNS. However, further analyses of our data suggest that high Tmax variability is masking Nrf2 activation in individual donors. Additionally, there may be sub-populations of responders, perhaps related to genetic polymorphisms in Nrf2.

Analysis of IL-6, IL-1ß and TNF-α production in monocytes isolated from multiple sclerosis patients treated with disease modifying drugs.

OBJECTIVE AND DESIGN: The etiology of multiple sclerosis (MS) is unknown, but blood derived monocytes/macrophages are believed to be involved in the pathogenesis through phagocytosis of myelin and production of inflammatory mediators. The objective of this study is to examine inflammatory cytokines that are present at elevated levels in active MS lesions to determine whether there are differences between classically stimulated monocytes isolated from healthy control (HC) and relapsing-remitting MS (RRMS) subjects taking disease modifying drugs (DMDs), including dimethyl fumarate (DMF). SUBJECTS: Thirty-nine veterans of the US Armed Forces were enrolled, 21 health controls (HC), and 18 with relapsing-remitting MS (RRMS), all taking DMDs. METHODS: Use ELISAs to measure production of IL-6, IL-1ß and TNF-α by LPS-stimulated peripheral monocytes. RESULTS: Activation of monocytes from MS subjects produced significantly more IL-6 than healthy controls (49531 ± 20795 vs 10526 ± 4845), and IL-6 production trended higher in MS subjects taking DMF than those taking other DMDs (72186.9 ± 35156.2 vs 32585.8 ± 17135.4). There were no significant differences in IL-1ß or TNF-α secretion. CONCLUSIONS: Our data suggest that not all DMDs may provide disease modification by suppressing monocyte/macrophage production of pro-inflammatory mediators.

Dimethyl fumarate activates the prostaglandin EP2 receptor and stimulates cAMP signaling in human peripheral blood mononuclear cells.

Dimethyl fumarate (DMF) was recently approved by the FDA for the treatment of relapsing remitting MS. The pathology of MS is a result of both immune dysregulation and oxidative stress induced damage, and DMF is believed to have therapeutic effects on both of these processes. However, the mechanisms of action of DMF are not fully understood. To determine if DMF is able to activate signaling cascades that affect immune dysregulation, we treated human peripheral blood mononuclear cells with DMF. We discovered that DMF stimulates cyclic adenosine monophosphate (cAMP) production after 1 min treatment in vitro. cAMP is a small molecule second messenger that has been shown to modulate immune response. Using pharmacological inhibitors, we determined that adenylyl cyclase mediates DMF induced cAMP production; DMF activated the prostaglandin EP2 receptor to produce cAMP. This response was not due to increased endogenous production of prostaglandin E2 (PGE2), but was enhanced by addition of exogenous PGE2. Furthermore, we determined that the bioactive metabolite of DMF, monomethyl fumarate (MMF), also stimulates cAMP production. These novel findings suggest that DMF may provide protection against MS by inhibiting immune cell function via the cAMP signaling pathway.

α-Tocopherol (vitamin E) stimulates cyclic AMP production in human peripheral mononuclear cells and alters immune function.

α-Tocopherol, the most biologically active member of the vitamin E family of fat soluble compounds, exhibits both antioxidant and anti-inflammatory properties. However, its mechanisms of action are not fully understood. Here, we show that, unlike other antioxidants, α-tocopherol stimulates the production of cyclic adenosine monophosphate (cAMP). Inhibitor studies demonstrate that the prostaglandin EP2 and EP4 receptors and adenylyl cyclases mediate the effects of α-tocopherol on cAMP production. Additionally, we show that α-tocopherol attenuates pro-inflammatory cytokine and chemokine production. This study provides novel evidence that α-tocopherol stimulates cAMP signaling, suggesting a mechanism of action for the immunomodulatory effects of vitamin E.

Lipoic acid stimulates cAMP production via G protein-coupled receptor-dependent and -independent mechanisms.

Lipoic acid (LA) is a naturally occurring fatty acid that exhibits anti-oxidant and anti-inflammatory properties and is being pursued as a therapeutic for many diseases including multiple sclerosis, diabetic polyneuropathy and Alzheimer's disease. We previously reported on the novel finding that racemic LA (50:50 mixture of R-LA and S-LA) stimulates cAMP production, activates prostanoid EP2 and EP4 receptors and adenylyl cyclases (AC), and suppresses activation and cytotoxicity in NK cells. In this study, we present evidence that furthers our understanding of the mechanisms of action of LA. Using various LA derivatives, such as dihydrolipoic acid (DHLA), S,S-dimethyl lipoic acid (DMLA) and lipoamide (LPM), we discovered that only LA is capable of stimulating cAMP production in NK cells. Furthermore, there is no difference in cAMP production after stimulation with either R-LA, S-LA or racemic LA. Competition and synergistic studies indicate that LA may also activate AC independent of the EP2 and EP4 receptors. Pretreatment of PBMCs with KH7 (a specific peptide inhibitor of soluble AC) and the calcium inhibitor (Bapta) prior to LA treatment resulted in reduced cAMP levels, suggesting that soluble AC and calcium signaling mediate LA stimulation of cAMP production. In addition, pharmacological inhibitor studies demonstrate that LA also activates other G protein-coupled receptors, including histamine and adenosine but not the ß-adrenergic receptors. These novel findings provide information to better understand the mechanisms of action of LA, which can help facilitate the use of LA as a therapeutic for various diseases.

Lipoic acid attenuates inflammation via cAMP and protein kinase A signaling.

BACKGROUND: Abnormal regulation of the inflammatory response is an important component of diseases such as diabetes, Alzheimer's disease and multiple sclerosis (MS). Lipoic acid (LA) has been shown to have antioxidant and anti-inflammatory properties and is being pursued as a therapy for these diseases. We first reported that LA stimulates cAMP production via activation of G-protein coupled receptors and adenylyl cyclases. LA also suppressed NK cell activation and cytotoxicity. In this study we present evidence supporting the hypothesis that the anti-inflammatory properties of LA are mediated by the cAMP/PKA signaling cascade. Additionally, we show that LA oral administration elevates cAMP levels in MS subjects. METHODOLOGY/PRINCIPAL FINDINGS: We determined the effects of LA on IL-6, IL-17 and IL-10 secretion using ELISAs. Treatment with 50 µg/ml and 100 µg/ml LA significantly reduced IL-6 levels by 19 and 34%, respectively, in T cell enriched PBMCs. IL-17 levels were also reduced by 35 and 50%, respectively. Though not significant, LA appeared to have a biphasic effect on IL-10 production. Thymidine incorporation studies showed LA inhibited T cell proliferation by 90%. T-cell activation was reduced by 50% as measured by IL-2 secretion. Western blot analysis showed that LA treatment increased phosphorylation of Lck, a downstream effector of protein kinase A. Pretreatment with a peptide inhibitor of PKA, PKI, blocked LA inhibition of IL-2 and IFN gamma production, indicating that PKA mediates these responses. Oral administration of 1200 mg LA to MS subjects resulted in increased cAMP levels in PBMCs four hours after ingestion. Average cAMP levels in 20 subjects were 43% higher than baseline. CONCLUSIONS/SIGNIFICANCE: Oral administration of LA in vivo resulted in significant increases in cAMP concentration. The anti-inflammatory effects of LA are mediated in part by the cAMP/PKA signaling cascade. These novel findings enhance our understanding of the mechanisms of action of LA.

Lipoic acid: a novel therapeutic approach for multiple sclerosis and other chronic inflammatory diseases of the CNS.

The naturally occurring antioxidant lipoic acid (LA) was first described as an essential cofactor for the conversion of pyruvate to Acetyl-CoA, a critical step in respiration. LA is now recognized as a compound that has many biological functions. Along with its reduced form dihydrolipoic acid (DHLA), LA reduces and recycles cellular antioxidants such as glutathione, and chelates zinc, copper and other transition metal ions in addition to heavy metals. LA can also act as a scavenger of reactive oxygen and nitrogen species. By acting as an insulin mimetic agent, LA stimulates glucose uptake in many different cell types and can also modulate insulin signaling. The p38 and ERK MAP kinase pathways, AKT and NFkappaB are all regulated by LA. In addition, LA activates the prostaglandin EP2 and EP4 receptors to stimulate the production of the small molecule cyclic adenosine 5' monophosphate (cAMP). These diverse actions suggest that LA may be therapeutically effective in treating oxidative stress associated diseases. This review discusses the known biochemical properties of LA, its antioxidant properties, its ability to modulate signal transduction pathways, and the recent progress made in the utilization of LA as a therapeutic alternative for multiple sclerosis, Alzheimer's disease and diabetic neuropathy.

Small heat shock proteins in smooth muscle.

The small heat shock proteins (HSPs) HSP20, HSP27 and alphaB-crystallin are chaperone proteins that are abundantly expressed in smooth muscles are important modulators of muscle contraction, cell migration and cell survival. This review focuses on factors regulating expression of small HSPs in smooth muscle, signaling pathways that regulate macromolecular structure and the biochemical and cellular functions of small HSPs. Cellular processes regulated by small HSPs include chaperoning denatured proteins, maintaining cellular redox state and modifying filamentous actin polymerization. These processes influence smooth muscle proliferation, cell migration, cell survival, muscle contraction and synthesis of signaling proteins. Understanding functions of small heat shock proteins is relevant to mechanisms of disease in which dysfunctional smooth muscle causes symptoms, or is a target of drug therapy. One example is that secreted HSP27 may be a useful marker of inflammation during atherogenesis. Another is that phosphorylated HSP20 which relaxes smooth muscle may prove to be highly relevant to treatment of hypertension, vasospasm, asthma, premature labor and overactive bladder. Because small HSPs also modulate smooth muscle proliferation and cell migration they may prove to be targets for developing effective, novel treatments of clinical problems arising from remodeling of smooth muscle in vascular, respiratory and urogenital systems.

Lipoic acid stimulates cAMP production via the EP2 and EP4 prostanoid receptors and inhibits IFN gamma synthesis and cellular cytotoxicity in NK cells.

The antioxidant lipoic acid (LA) treats and prevents the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In an effort to understand the therapeutic potential of LA in MS, we sought to define the cellular mechanisms that mediate the effects of LA on human natural killer (NK) cells, which are important in innate immunity as the first line of defense against invading pathogens and tumor cells. We discovered that LA stimulates cAMP production in NK cells in a dose-dependent manner. Studies using pharmacological inhibitors and receptor transfection experiments indicate that LA stimulates cAMP production via activation of the EP2 and EP4 prostanoid receptors and adenylyl cyclase. In addition, LA suppressed interleukin (IL)-12/IL-18 induced IFNgamma secretion and cytotoxicity in NK cells. These novel findings suggest that LA may inhibit NK cell function via the cAMP signaling pathway.

Overexpression of human Hsp27 inhibits serum-induced proliferation in airway smooth muscle myocytes and confers resistance to hydrogen peroxide cytotoxicity.

Airway smooth muscle (ASM) hypertrophy and hyperplasia are characteristics of asthma that lead to thickening of the airway wall and obstruction of airflow. Very little is known about mechanisms underlying ASM remodeling, but in vascular smooth muscle, it is known that progression of atherosclerosis depends on the balance of myocyte proliferation and cell death. Small heat shock protein 27 (Hsp27) is antiapoptotic in nonmuscle cells, but its role in ASM cell survival is unknown. Our hypothesis was that phosphorylation of Hsp27 may regulate airway remodeling by modifying proliferation, cell survival, or both. To test this hypothesis, adenoviral vectors were used to overexpress human Hsp27 in ASM cells. Cells were infected with empty vector (Ad5) or wild-type Hsp27 (AdHsp27 WT), and proliferation and death were assessed. Overexpressing Hsp27 WT caused a 50% reduction in serum-induced proliferation and increased cell survival after exposure to 100 microM hydrogen peroxide (H(2)O(2)) compared with mock-infected controls. Overexpression studies utilizing an S15A, S78A, and S82A non-phosphorylation mutant (AdHsp27 3A) and an S15D, S78D, and S82D pseudo-phosphorylation mutant (AdHsp27 3D) showed phosphorylation of Hsp27 was necessary for regulation of ASM proliferation, but not survival. Hsp27 provided protection against H(2)O(2)-induced cytotoxicity by upregulating cellular glutathione levels and preventing necrotic cell death, but not apoptotic cell death. The results support the notion that ASM cells can be stimulated to undergo proliferation and death and that Hsp27 may regulate these processes, thereby contributing to airway remodeling in asthmatics.

Synthesis of immune modulators by smooth muscles.

The primary function of smooth muscle cells is to contract and alter the stiffness or diameter of hollow organs such as blood vessels, the airways and the gastrointestinal and urogenital tracts. In addition to purely structural functions, smooth muscle cells may play important metabolic roles, particularly in various inflammatory responses. In cell culture, these cells have been shown to be metabolically dynamic, synthesizing and secreting extracellular matrix proteins, glycosaminoglycans and a wide variety of cell-cell signaling proteins, such as interleukins, chemokines and peptide growth factors. Secreted cell signaling proteins participate in the inflammatory response of smooth muscle-containing organs, and some can also stimulate smooth muscle migration, proliferation and contraction. The cellular signaling pathways controlling synthesis of these signaling proteins are similar to those used by cells mediating innate immunity and may contribute to pathogenesis of diverse diseases including atherosclerosis, asthma, inflammatory bowel diseases and preterm labor. Appreciating the role of smooth muscle cells in these diseases may lead to better understanding of the beneficial effects of anti-inflammatory drugs as well as identification of new targets for anti-inflammatory therapy.

Inflammatory gene expression by human colonic smooth muscle cells.

Intestinal mucosal cells and invading leukocytes produce inappropriate levels of cytokines and chemokines in human colitis. However, smooth muscle cells of the airway and vasculature also synthesize cytokines and chemokines. To determine whether human colonic myocytes can synthesize proinflammatory mediators, strips of circular smooth muscle and smooth muscle cells were isolated from human colon. Myocytes and muscle strips were stimulated with 10 ng/ml of IL-1beta, TNF-alpha, and IFN-gamma, respectively. Expression of mRNA for IL-1beta, IL-6, IL-8, and cyclooxygenase-2 (COX-2) was induced within 2 h and continued to increase for 8-12 h. Regulated on activation, normal T cell-expressed and -secreted (RANTES) mRNA expression was slower, appearing at 8 h and increasing linearly through 20 h. Expression of all five mRNAs was inhibited by 0.1 microM MG-132, a proteosome inhibitor that blocks NF-kappaB activation. Expression of IL-1beta, IL-6, IL-8, and COX-2 mRNA was reduced by 30 microM PP1, an Src family tyrosine kinase inhibitor, and by 25 microM SB-203580, a p38 MAPK inhibitor. MAPK/extracellular regulated kinase-1 inhibitor PD-98059 (25 microM) was much less effective. In conclusion, human colonic smooth muscle cells can synthesize and secrete interleukins (IL-1beta and IL-6) and chemokines (IL-8 and RANTES) and upregulate expression of COX-2. Regulation of cytokine, chemokine, and COX-2 mRNA depends on multiple signaling pathways, including Src-family kinases, extracellular regulated kinase, p38 MAPKs, and NF-kappaB. SB-203580 was a consistent, efficacious inhibitor of inflammatory gene expression, suggesting an important role of p38 MAPK in synthetic functions of human colonic smooth muscle.