Lipid Mediators of Inflammation PDF

Lipid Mediators of Inflammation Scientific Foundations 1, Biochemistry Learning Objectives Describe the synthesis of the major classes of eicosanoids derived from the w-6 fatty acid arachidonic acid—prostanoids, leukotrienes, and lipoxins—distinguishing between cyclooxygenase-...

Lipid Mediators of Inflammation Scientific Foundations 1, Biochemistry Learning Objectives Describe the synthesis of the major classes of eicosanoids derived from the w-6 fatty acid arachidonic acid—prostanoids, leukotrienes, and lipoxins—distinguishing between cyclooxygenase- and lipoxygenase-dependent pathways. Describe the synthesis of the major classes of lipid mediators derived from the w-3- polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Outline the mechanism of action and describe the primary physiological effects of the pro-inflammatory eicosanoids, as well as the lipoxins, epi-lipoxins, resolvins, protectins, and maresins. Identify the major molecular targets of pharmaceutical agents that inhibit the synthesis of lipid mediators of inflammation. Concept Statement Inflammation is a protective biological response that is initiated after infection or injury. It is essential for the elimination of the inciting stimulus. Chronic or inappropriate inflammation is associated with many pathological conditions such as asthma. Certain lipids derived from arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) act as autocrine and paracrine signaling molecules with both pro-inflammatory and anti-inflammatory effects. These molecules are critical to both healthy and pathological inflammatory states, and their synthesis is a frequent target of therapeutic intervention. Lipid mediators are involved in both initiation and resolution of inflammation! I. Essential Fatty Acids The lipid mediators of inflammation are derived primarily from three precursor poly- unsaturated fatty acids (PUFAs): arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). While these PUFAs are not themselves essential in the diet, their precursors are. Thus, they can be considered conditionally essential. The w-6 fatty acid linoleic acid and the w-3 fatty acid a-linolenic acid are essential in the diet. These fatty acids are found in seed oils such as sunflower, flax, and chia, while fish oil is rich in EPA and DHA. Humans encode the enzymatic machinery to synthesize arachidonic acid using linoleic acid as a precursor and to synthesize both EPA and DHA using a-linolenic acid as a precursor. 1 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones.  Omega 6 Omega 3 Linoleic Acid a-Linolenic Acid soybean, sunflower, flax and chia oils canola oils g-Linolenic Acid EPA evening primrose fish oil and barrage oils Arachidonic acid DHA meat fish and algae oil AA, DHA, and EPA are incorporated into the membrane phospholipids in many different cells and tissues. Thus, the substrates for generation of lipid mediators of inflammation are distributed all over the body, but not all tissues produce all lipid mediators! When looking at the complex synthesis pathways that produce mediators in each family, keep in mind that each tissue expresses only a subset of the enzymatic machinery. Furthermore, the lipid mediators transduce their signals via binding to specific G-protein- coupled receptors, which are also expressed in a tissue-specific manner. These lipid mediators are very short-lived, so they can only exert their effects locally via autocrine and paracrine signaling. II. Arachidonic Acid Pathways A. Overview AA is the precursor to a wide variety of lipid mediators, including those with pro- and anti- inflammatory effects and those involved in other physiological processes. The synthesis pathway begins with activation of phospholipase A2 (PLA2), which cleaves AA from a membrane phospholipid, and then proceeds down one of two pathways dependent on either cyclooxygenase or lipoxygenase. 2 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones.  Robbins and Cotran Pathologic Basis of Disease by Vinay Kumar; Abul K. Abbas; Jon C. Aster B. Phospholipase A2 Phospholipase enzymes cleave phospholipids at various positions. Phospholipase C, for example, cleaves PIP2 to generate PIP3 and diacylglycerol (DAG). Phospholipase A1 and A2, on the other hand, cleave the bond between the lipid and the glycerol backbone, generating free fatty acid and lysophospholipid. AA is normally found in the R2 position, so PLA2 will be of most interest. Activation of PLA2 is a multistep process which actually begins with activation of PLC in response to extracellular signals. PIP2 is hydrolyzed by PLC to form the secondary messengers IP3 and DAG. IP3 opens up the Ca2+ ion channel in the perinuclear membrane of the ER, which 3 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones. increases the intracellular Ca2+ concentration. The DAG and Ca2+ together activate protein kinase C (PKC), which activates MAPK. MAPK phosphorylates cPLA2. Ca2+ ions promote association of cPLA2 with the membrane; however, the catalytic domain is not properly oriented. Phosphorylation by MAPK in the flexible linker of the cPLA2 induces optimal conformation of the catalytic domain, allowing it to cleave the phospholipid and release AA. Int. J. Mol. Sci. 2023, 24(2), 135 Clinical Correlation: PLA2 is a frequent target of pharmaceutical intervention. Corticosteroids are used to reduce the total activity of PLA2 and thus the pool of AA. In general, steroids exert their effects by binding to their receptors which then act as transcription factors. Corticosteroids increase transcription of the gene for lipocortin, an endogenous inhibitor of PLA2. They also decrease transcription of the gene for PLA2. Because they work at the level of protein expression, they are relatively slow to take effect. Corticosteroids are prescribed for pathological inflammatory conditions from asthma to poison ivy. C. Cyclooxygenase Pathways: The Prostanoids Synthesis The prostanoids include prostaglandins, prostacyclins, and thromboxane, all of which depend on the enzyme cyclooxygenase (COX). COX carries out bisoxygenation and cyclization of AA, followed by reduction at the 15 position to generate the common prostanoid intermediate PGH2. Thus, COX gives the prostanoids their characteristic cyclic structure. Tissue-specific 4 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones. isomerases, peroxidases, etc., will then generate the active members of the family, prostaglandins (PGD2, PGE2, PGF2a), prostacyclin (PGI2), and thromboxane A2 (TXA2). Degenerative Neurological and Neuromuscular Disease 2020:10 1–13 Physiological Effects The effects of these mediators are many and varied. PGE2 and PGI2 are classic pro-inflammatory lipids mediators, bringing about vasodilation, edema, and increased vascular permeability. While they do not directly cause pain, they reduce the threshold of nociceptor sensory neurons, bringing about hyperalgesia, so their release can increase the sensation of pain. PGE2 is also highly pyretic. PGD2, on the other hand, promotes the resolution of inflammation, so it can temper the impact of the inflammatory prostanoids. Perhaps counterintuitively, PGE2 and PGI2 are also critical for gastric protection, where, among other things, they stimulate mucus and bicarbonate secretion. The prostanoids play critical roles in the menstrual cycle and pregnancy. PGF2a, for example, promotes uterine smooth muscle contraction and vasoconstriction of the endometrial vessels 5 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones. during menstruation. Both PGE2 and PGF2a are important for labor, with exogenous PGF2a sometimes used to induce labor. As is implied by its name, TXA2 promotes thrombosis by stimulating vasoconstriction and platelet aggregation. The activity is directly countered by PGI2, which in addition to vasodilation, promotes platelet disaggregation. Thus, the balance between these two is critical for cardiovascular health. Cyclooxygenase There are two main isoforms of COX, COX-1 and COX-2. In general, COX-1 is constitutively expressed in many tissues and performs normal housekeeping functions, while COX-2 expression is more limited in its tissue distribution and is rapidly induced in response to inflammatory stimuli. Notably, individual prostanoids are not limited to synthesis by a single isoform of COX. Instead they are synthesized for different purposes in different tissues. PGE2 and PGI2, for example, play both housekeeping and inflammatory roles. SCIENCE 2012 Vol 336 (6087) 1386-1387 Clinical Correlation: Both COX-1 and COX-2 are the targets of nonsteroidal anti-inflammatory drugs (NSAIDS), so-called to distinguish them from the corticosteroids that are also used combat chronic inflammation. The non-selective NSAIDs act on both COX-1 and COX-2. They include common over-the-counter medications such as ibuprofen and naproxen, which act as reversible inhibitors of both enzymes, and aspirin (acetylsalicylic acid). While aspirin—and its natural source willow bark—can be considered an ancient medicine, its mechanism and physiological target have only been elucidated relatively recently. Dr. John Robert Vane was awarded the Nobel Prize in Physiology and Medicine in 1982 for his discovery (10 years earlier) 6 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones. that aspirin reduced prostaglandin and thromboxane synthesis. We now know that aspirin acetylates both COX-1 and COX-2, resulting in irreversible inhibition of COX-1 and modification of COX-2 activity. More on that below. Several selective inhibitors of COX-2 (coxibs) have been developed. However, these were found to increase the risk of stroke, myocardial infarction, thrombosis, and other CV-related events. This is likely due to disruption of the balance between pro-thrombotic TXA2 and anti- thrombotic PGI2. That said, and least one coxib for which side effects are relatively mild is still available by prescription, and more recently some of the non-selective NSAIDs have also been shown to increase the risk of CV events. That’s probably enough on this topic for a non- pharmacology lecture. D. Lipoxygenase Pathways: Leukotrienes, Lipoxins, Epi-Lipoxins Leukotriene Synthesis As with COX, lipoxygenase (LOX) has several isoforms. Unlike COX, the different isoforms of LOX are more closely associated with distinct families of mediators and are more tissue restricted in their expression. LOX-5 is expressed primarily in leukocytes, and it is responsible for production of the leukotrienes, powerful mediators of the allergic response and the so-called slow reacting substances of anaphylaxis. Upon activation by Ca2+, phosphorylation, and the accessory protein FLAP, LOX-5 converts AA into the common intermediate 5-HPETE and then LTA4. This is then converted either to the active compound LTB4, or to one of the active “peptide” or “cysteinyl” LTs through conjugation to a glutathionyl group at C-6 to form LTC4, LTD4, and LTE4. 7 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones.  Atherosclerosis 224 (2012) 129-135 Leukotriene Physiological Effects As mentioned above, LTs are powerful mediators of the allergic response. LTB4 is a chemoattractant for neutrophils and eosinophils. The cysteinyl LTs bind with differing levels of affinity to two seven-transmembrane receptors, CysLT1 and CysLT2. Binding and activation of CysLT1 leads to bronchoconstriction, mucus secretion, and edema in the airways, while CysLT2 activation leads to inflammation, vascular permeability, and lung fibrosis. Clinical Correlation: LT receptor antagonists (lukasts) have been developed to counteract this effect and are used in patients with asthma, allergic rhinitis, and chronic obstructive pulmonary disease. Lipoxin and Epi-Lipoxin Synthesis The lipoxygenase isoforms LOX-15 and LOX-12 catalyze the formation of the anti-inflammatory lipoxins LXA4 and LXB4. The synthesis of both lipoxins takes places through an unusual transcellular process, in which an intermediate is released from one cell and then taken up by a different cell to complete synthesis. In one pathway, LOX-15 catalyzes formation of the intermediate 5(6) epoxytetraene in eosinophils, monocytes, and epithelial cells, and this intermediate is further processed by LOX-5 in monocytes and neutrophils. Alternatively, LTA4 released from leukocytes can be processed by LOX-12 in platelets. Synthesis of the epi-lipoxins (15-epi-lipoxin A4 and B4) is catalyzed by COX-2 that has undergone aspirin-dependent 8 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones. acetylation. Thus, aspirin decreases inflammation not only by inhibiting synthesis of pro- inflammatory mediators, but also by stimulating synthesis of anti-inflammatory mediators. Molecular Interventions 2006 6(4):199-207 6(4):199-207 Lipoxin and Epi-Lipoxin Physiological Effects LX induce the resolution phase of the inflammatory response by promoting ingestion of apoptotic neutrophil fragments by macrophage, and stimulating production of anti- inflammatory cytokines. Epi-LX perform the same functions as LX, and also induce vasorelaxation, increase synthesis of NO, and inhibit pro-inflammatory cytokine production. III. w-3 Fatty Acid Pathways A. Overview The essential w-3 PUFA a-linolenic acid can be converted to EPA and DHA by enzymes expressed in many human tissues. These in turn serve as precursors for synthesis of the anti- inflammatory lipid mediators known as resolvins, protections, and maresins. Much less is known about the synthesis and physiological effects of these molecules, so this section will be relatively brief. However, research in this area is very active and forms the basis for many dietary and supplement recommendations. All physicians should therefore have some familiarity with it. 9 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones. EPA is a substrate for both COX isoforms as well as LOX-5, which can convert it to the 3-series prostanoids and the 5-series leukotrienes, respectively. The molecules can bind to the same receptors as their 2- and 4- series counterparts described above, but they are only weakly inflammatory. Thus, they serve as competitive inhibitors with the overall effect of decreasing the magnitude of the inflammatory response. EPA can also be converted to the E-series resolvins by cytochrome p450 or acetyl-COX-2, providing another example of the anti- inflammatory impact of aspirin. The pathways required for DHA conversion to D-series resolvins, protectins, and maresins are quite complex (and in the case of maresins, still theoretical). They each involve sequential activity of multiple LOX isoforms and/or acetyl-COX-2 as well as transcellular synthesis. Eicosapentaenoic Acid Docosahexaenoic Acid COX1 and Acetyl-COX-2* Lipoxygenase LOX-5 COX2 or Cyt p450 Acetyl-COX-2 3 Series 5 Series E-Series D-Series Prostanoids Leukotrienes Resolvins Resolvins Protectins Maresins B. Physiological Effects The resolvins temper the severity of the inflammatory response and promote resolution of inflammation by a variety of mechanisms. They reduce neutrophil and dendritic cell accumulation by blocking their migration and enhancing clearance. The also reduce the production of reactive oxygen species during the neutrophil oxidative burst and the synthesis of pro-inflammatory cytokines. As with the lipoxins, they stimulate ingestion of apoptotic fragments by macrophage as part of inflammation resolution. Protectins appear to share many of the same features, and they also reduce signaling through toll-like receptors and T-cell migration. Maresins are the most recently identified anti-inflammatory mediators. As mentioned above, their synthetic pathways have not been fully elucidated. However, they appear to carry out many of the same anti-inflammatory processes of the related resolvins and protectins, blocking infiltration of neutrophils and stimulating macrophage phagocytic activity. Summary 10 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones.  Lipid mediators of inflammation are fast acting autocrine and paracrine signaling molecules whose synthesis and activity are tissue specific. The w-6 fatty acid arachidonic acid is the precursor for many pro- and anti-inflammatory molecules, including the prostanoids, leukotrienes, lipoxins and epi-lipoxins. Synthesis of the prostanoids (prostaglandins, prostacyclins, and thromboxane) is dependent on COX-1 or COX-2. Prostaglandins and prostacyclins are usually pro- inflammatory. Synthesis of leukotrienes is dependent on LOX-5. Leukotrienes are usually pro- inflammatory LOX-12, LOX-15, and acetyl-COX-2 promote synthesis of the anti-inflammatory lipoxins and epi-lipoxins. The w-3 fatty acids EPA and DHA can be converted to E- and D-series resolvins, protectins, and maresins, which all have anti-inflammatory properties. Acknowledgement: This hand-out relies heavily on the article “Lipid Mediators in Inflammation” by Melanie Bennett and Derek Gilroy (Microbiol Spectrum 4(6):MCHD-0025-2016). This is a really thorough review of the topic, and I recommend it for anyone who wants to learn more (a lot more) about these classes of molecules. Be forewarned, it’s 21 pages long and there are no pictures, but it is very clearly written. 11 © Dr. Jessica M. Jones. This material may not be re-printed or shared without explicit permission of Dr. Jessica M. Jones.