Lipoic Acid Attenuates Inflammation via cAMP and Protein Kinase A Signaling

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.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.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

A-Kinase Anchoring in Dendritic Cells Is Required for Antigen Presentation

BACKGROUND: Dendritic cells (DC) are the most potent antigen presenting cells (APC) of the immune system. Prostaglandin E(2), cyclic AMP, and protein kinase A (PKA) have all been shown to regulate DC maturation and activity. In other cells, the ability of these molecules to convey their signals has been shown to be dependent on A-kinase anchoring proteins (AKAPs). Here we present evidence for the existence and functional importance of AKAPs in human DC. METHODOLOGY/PRINCIPAL FINDINGS: Using immunofluorescence and/or western analyses we identify AKAP79, AKAP149, AKAP95, AKAP LBC and Ezrin. We also demonstrate by western analysis that expression of AKAP79, AKAP149 and RII are upregulated with DC differentiation and maturation. We establish the functional importance of PKA anchoring in multiple aspects of DC biology using the anchoring inhibitor peptides Ht31 and AKAP-IS. Incubation of protein or peptide antigen loaded DC with Ht31 or AKAP-IS results in a 30-50% decrease in antigen presentation as measured by IFN-gamma production from antigen specific CD4(+) T cells. Incubation of LPS treated DC with Ht31 results in 80% inhibition of TNF-alpha and IL-10 production. Ht31 slightly decreases the expression of CD18 and CD11a and CD11b, slightly increases the basal expression of CD83, dramatically decreases the LPS stimulated expression of CD40, CD80 and CD83, and significantly increases the expression of the chemokine receptor CCR7. CONCLUSIONS: These experiments represent the first evidence for the functional importance of PKA anchoring in multiple aspects of DC biology

Treatment with LA attenuates T cell proliferation and activation.

<p>(A) T cell enriched PBMCs were pretreated with LA prior to stimulation with anti-CD3/CD28 for 72 hours. Proliferation was measured using 3H-Thymidine incorporation. N = 3. *indicates statistical significance compared to anti-CD3/CD28 control (<i>p</i><0.05). (B) T cell enriched PBMCs were stimulated in triplicate with anti-CD3 (4 µg/ml, OKT3) and soluble anti-CD28 (5 µg/ml, BD Pharmingen) in the presence or absence of 50 µg/ml LA (1 minute pretreatment). Cells were then incubated for 6 hours at 37°C, 5% CO2. Supernatants were collected and IL-2 levels were analyzed by ELISA (R&D Systems, Minneapolis, MN). N = 3. *indicates statistical significance compared to anti-CD3/CD28 control (<i>p</i><0.05).</p

Schematic illustration of the putative LA signaling events.

<p>LA binds to the EP2 and EP4 receptors to activate the Gs subunit of the trimeric G-proteins. Gs activates adenylyl cyclase (AC), which generates cAMP from ATP. AC is degraded by phosphodiesterases (PDEs) into AMP and Pi. cAMP activates the PKA signaling cascade leading to inhibition of immune cell function. PKI acts as a pseudosubstrate to inhibit PKA signaling.</p