12.1 Introduction

inflammation in chronic conditions (for reviews see1–4). Many of the mechanisms of phytochemical suppression of inflammation involve blocking NF-kB- induced gene expression of proinflammatory mediators. Examples include plant polyphenols or flavonols. Other actions of phytochemicals involve stimulation of the transcription factors, peroxisome proliferator-activated receptor g (PPARg) and liver-X-receptor (LXR), which indirectly repress NF-kB, such as plant-derived fatty acids or plant sterols.

This chapter focuses mainly on the influence of some of the more common natural products on transcription factors, which are the most promising targets for designing new active drugs against inflammation. Alternatively current herbal therapeutic remedies have been recognized for centuries. Ultimately the question may revolve around the choice between dampening inflammation with long-term, but possibly less potent dietary ligands versus more potent synthetic analogs under acute conditions, but which may have uncertain toxicity.

12.2 Overview of the Inflammatory Cascade

The process of inflammation involved in all tissues can be initiated by cells such as macrophages and dendritic cells. These cells can undergo activation, whereupon they release inflammatory mediators responsible for propagating an inflammatory cascade. TNFa and interleukin-1 (IL-1) are cytokines produced primarily in macrophages, to induce fever, production of more cytokines, endothelial regulation, chemotaxis and leukocyte adherence.

If inflammatory mechanisms are not downregulated appropriately, conditions such as rheumatoid arthritis may develop, among other conditions. Production of TNFa, IL-6, IL-8, histamine and other inflammatory mediators by activated mast cells are believed to drive synovitis in rheumatoid arthritis.

It is now known that innate immune mechanisms become amplified in acute coronary syndromes. In atherosclerotic lesions, platelets become incorporated into growing thrombi, which results in the surface expression of P-selectin. This binds to leukocytes, thereby mediating monocyte and granulocyte activation and thrombus adhesion. Trapped platelets and leucocytes within a thrombus secrete RANTES (regulated upon activation, normal T-cell expressed and secreted) and other inflammatory mediators resulting in activation of other immune pathways and thrombotic mediators, such as the release of TNFa and tissue factors, thereby escalating tissue injury.

Many neurodegenerative conditions also involve inflammatory mechanisms that may be causative in etiology or amplify the condition, such as multiple sclerosis. Multiple sclerosis is known to involve T-cell entry into the brain across the blood-brain barrier. T-cells then recognize myelin as an antigen and trigger inflammatory pathways and antibodies toward myelin. This stimulates increased levels of cytokines, swelling, activation of macrophages and microglia and more pathology.

Alzheimer’s disease also seems to have an inflammatory component, whether it is a primary event in its etiology or a secondary event, involved in a later

process.5 Alzheimer’s disease also involves the accumulation of amyloid beta (Ab) peptide deposits and phosphorylated tau, which appear to promote neurofibrillary tangles and plaques in the brain. However, bystander damage from inflammation appears to at least exacerbate pathology, as the upregulation of complement, cytokines, acute phase reactants and other inflammatory mediators are present in a ected tissues.

12.3 Overview of NF-jB

When immune cells are activated by cytokines such as TNFa, IL-1 or toll-like receptor activators binding to their receptor targets, NF-kB is also activated in a complex set of pathways. NF-kB is a ubiquitous transcription factor that controls the expression of genes involved in inflammatory responses. Increased over-activation of NF-kB may cause inflammatory and autoimmune diseases, or other chronic conditions. Five mammalian NF-kB family members have been identified:

NF-kB1 (also known as p50)

NF-kB2 (also known as p52)

RelA (also known as p65)

RelB

RelC

They all share a highly conserved Rel homology domain, responsible for their dimerization and binding to DNA and IkB (inhibitor of NF-kB). The transcription factor NF-kB works only when two members form a dimer. The most abundant activated form consists of a p50 or p52 subunit and a p65 subunit.

As shown in Figure 12.1, NF-kB is located in the cytosol, in an inactive state, complexed with the inhibitory protein, IkBa. Extracellular signals such as cytokines activate the enzyme IkB kinase (IKK). IKK then phosphorylates the IkBa protein, which becomes ubiquitinated. This results in the dissociation of IkBa from NF-kB and degradation of IkBa by the proteosome. The activated NF-kB is then translocated into the nucleus, where it binds to response elements in the promoter region of DNA in order to initiate transcription and be translated into proteins that magnify the inflammatory response.

The surface receptors (with associated ligands) that activate NF-kB are: RANK (Receptor activator of NF-kB, found on the surface of stromal cells, osteoblasts, T-cells and dendritic cells), TNFR (tumor necrosis factor receptor or CD120), lyphotoxin beta receptor, BAFF-R (B-cell activation receptor or CD40), tumor necrosis factor receptor superfamily, member 8 (TNFRSF8 or CD30, found on activated B or T-cells). In addition, bacterial products activate NF-kB, through toll-like receptors, such as TLR4, which is activated by LPS in Gram-negative bacteria. IL-1R interleukin-1 receptor (IL-1R) also activates NF-kB through the binding of its ligand, IL-1.

Figure 12.1 Overview of NF-kB signal transduction pathway.

12.4 PPARc and LXRs Regulate NF-jB

NF-kB can be negatively regulated by PPARg, LXR, glucocorticoid receptors, estrogen receptors, progesterone receptors and the androgen receptor. PPARg and LXR may present desirable strategies for altering NF-kB and the inflammatory cascade, since nearly all cells have a form of PPAR and LXR.

This chapter focuses on direct NF-kB inhibitory mechanisms (and indirect PPAR agonist–NF-kB inhibitory pathways) as having the potential for limiting inflammatory pathways, while avoiding the profound and undesirable e ects of hormones such as glucocorticoids. For instance, glucocorticoids not only directly inhibit NF-kB pathways, but also bind to glucocorticoid receptors that produce undesirable side-e ects, such as: suppression of calcium absorption in bone, hyperglycemia, muscle weakness and pubertal delay.

Similarly, steroid hormones such as estrogen inhibit NF-kB pathways, but also have multiple undesirable e ects, such as mitogenic e ects and the potential for tumor proliferation. PPAR agonists, in contrast, may be relatively benign.

PPARg is a transcription factor, which, when bound to the retinoic acid receptor (RXR), promotes transcription of proteins involved in fatty acid metabolism. It is activated by ligands such as fatty acids, oxLDL and thiazolidinediones. However, with PPARg a trans-repression mechanism occurs, such that when PPARg is inhibited NF-kB is activated promoting inflammation. Conversely, when PPARg is activated, NF-kB and hence inflammation are

inhibited. Thiazolidinediones are common PPARg agonists used in diabetes therapy, insulin resistance and obesity.

The three PPAR subtypes, a, g and d, regulate di erent lipoproteins and lipid homeostasis depending on the tissue. PPARa potentiates fatty-acid catabolism in the liver. However, PPARg is involved in adipocyte di erentiation and insulin sensitizing. PPARg also regulates insulin responsive genes involved in glucose production, transport and utilization. PPARa is expressed in liver, kidney, heart, muscle and adipose tissue. PPARb and PPARd are expressed in high levels in brain, adipose tissue and skin. PPARg is expressed in all tissues.

In macrophages, PPARg is believed to coordinate a complex response to oxLDL that involves increased scavenging ability and increased lipid e ux. One important role of PPARg in macrophages may be the removal of oxLDL from the artery wall and promoting cholesterol e ux through upregulation of a cholesterol transport pathway.6 Activation of PPARg reduces blood levels of triglycerides. Therefore inhibition of PPARg may result in elevated cholesterol and triglycerides while NF-kB-mediated inflammatory pathways are increased.

Another transcription factor involved in NF-kB regulation is LXR, so named because it is present in high levels in the liver, but is ubiquitous in all tissues. LXR and PPAR are nuclear receptor proteins of the same family, which, when activated by their ligands, become transcription factors for metabolic pathways. Similar to PPAR, LXRs are important regulators of cholesterol, fatty-acid metabolism and glucose homeostasis.7 Their activating ligands consist of oxysterols. LXR binds to RXR before transcription can occur. In addition, LXR acts as a trans-repressor of NF-kB analogous to PPAR.7 LXR agonists also inhibit expression of the protein products of NF-kB, such as COX-2, IL-1b, iNOS and TNFa. Therefore activators of LXR and PPAR also inhibit NF-kB-mediated inflammatory pathways. However, PPAR is more involved in fatty-acid metabolism, whereas LXR is more involved in cholesterol and oxysterol metabolism.

PPARg and LXR are of particular relevance to inflammatory pathways in macrophages. Since macrophages (and microglia) are primary generators of inflammatory cytokines and chronic immune responses, the modifying e ects of the PPARg and LXR transcription factors can dramatically a ect the immune response. For instance, in atherosclerotic lesions, macrophages are recruited to atherosclerotic plaques. If inhibition of PPARg or LXR occurs, lipid and cholesterol homeostasis is disturbed within the macrophage. This leads to inhibited e ux of lipid and cholesterol from the cell, in the form of HDL (in order for transport to the liver). This consequently results in the conversion of macrophages into foam cells, which are unable to phagocytize oxidized cholesterol properly. This also leads to the activation of NF-kB with increased local inflammation and pathology during a cardiovascular incident.8–11 Therefore, the inhibition of PPARg or LXR within macrophages means that they are not only failing to phagocytize apoptotic debris, pathogens or oxLDL, but they are promoting increased inflammation, oxidative stress and pathology.

This is why synthetic agonists of PPARg, such as thiazolidinediones, decrease cytokine production in activated macrophages and provide beneficial