Round 36: Systems Approach with Inflammatory Exudates – A Novel Genus of Anti-Inflammatory & Pro-Resolving Mediators

By: Charles N. Serhan, MD
Director & Professor, Center for Experimental Therapeutics, BWH
Professor, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine

Disclosures:

From our basic work supported by NIH, NIGMS, NIDDK and Specialized Center supported by NIDCR, we have captured IP, the intellectual property, that’s been licensed for clinical development by Bayer Health Care. I am a co-founder of Resolvyx Pharmaceuticals who also has permission to bring some of these molecules to the clinic.

Release Date: January 4, 2012
Expiration Date: October 31, 2013

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Introduction

The Center of Experimental Therapeutics is doing work on a novel genus of anti-inflammatory and pro-resolving mediators.

Above is an artist’s rendition of the battle wagered in acute inflammation/host defense. The surgeon’s cut, or a break in the barrier of the skin, with the invasion of microbes is illustrated. This is the milieu for the diathesis of lymphocytes. The signs of acute inflammation have been known since antiquity.  The upper right-hand corner of the figure shows the Egyptian hieroglyphics of the acute signs of inflammation. The unwanted side effect of this host defense is the acute inflammatory response. Current pharmacology has the ability to block the receptor antagonists or inhibitors of pro-inflammatory pathways. The notion has been that there are too many pro-inflammatory mediators; however, there’s an endogenous turn-on of termination signals that involves small chemical mediators and these new pro-resolving lipid mediators are actually useful templates for thinking about novel therapeutics.

Objectives

  • Determine which endogenous mediators control the magnitude and duration of an innate response
  • Discuss the impact of aspirin in the system
  • Identify key points of the structural elucidation of novel specialized chemical mediators, and the formation and action of resolvins and protectins
  • Discuss new work on microfluidic chambers that enables functional decoding metabolomics on a single-cell level

What endogenous mediators control the magnitude and duration of an innate response? 

The ideal outcome of inflammation is complete resolution. When headed towards acute inflammation, prostaglandins and leukotrienes are generated temporally and they are separated from the lipoxins. The move to chronic inflammation is generally thought to have an excess of prostaglandins and leukotrienes.

There is a lot of interest in inflammation because many nonclassic, widely occurring diseases in the population are thought to have an etiology based in aberrant inflammatory responses or uncontrolled inflammation. This includes asthma, cardiovascular disease, periodontal disease, arthritis and maybe even the course of aging. Acute inflammation could go through an abscess to healing, and because the lipoxins were temporally dissociated, questions about specialized chemical mediators that can handle this, and the resolvins and protectins started to emerge.

New genus of anti-inflammatory & pro-resolving lipid mediators

Lipid mediators switch class as they go from acute inflammation in the model system to complete resolution and homeostasis. Prostaglandins E2 and D2 play a very important role in stimulating the turning on of specialized chemical mediators.  Using the mouse model with fixed time zero there are going to be ideal definitions of self-limited or resolving systems and edema formation, followed by neutrophilic entry. The neutrophils decrease, and then the non-phlogistic infiltration of mononuclear cells and macrophages occur.  During the decrease in neutrophils, the first resolvins and protectins appear. Resolvins and protectins belong to a larger genus of molecules that can dampen acute inflammation and promote resolution.  Each of these series, the E-series resolvins and D-series resolvins, are unique chemical structures.

Neutrophils swarm rapidly, like bees, and inflammation happens very, very quickly.  For example, an instigator such as a laser zap can cause a principle chemoattractant of leukotriene D4 for leukocytes.  They move along a gradient in leukotriene D4 and rapidly congregate. There are endogenous mechanisms that could control this swarming.  The general theory has been that neutrophils activate and release their armamentaria to extracellular milieu. They can invoke tissue damage and injury, serving as sort of trigger-happy cells releasing this noxious material, hydrolytic enzymes, reactive oxygen species that then propagate inflammation. This is a reaction that does not dissipate passively.  Resolution in an ideal setting is an active process; it’s a biochemically active process that’s turned on and temporally orchestrated.

Murine air pouch

The murine air pouch is an air pouch that can be used on rats and the back of mice to study the initiation process, exudate formation and capture the histology because resolution is not a new concept.  Pathologists know when disease resolves by looking at tissue.

Within the milieu of a self-limited acute inflammatory response, the green area in the figure (right) is neutrophilic entry in the air pouches in response to TNF-alpha. The prostaglandin levels go up and then down and dissipate in this period.  Leukotriene D4 levels are up and then down during resolution.  Surprisingly, lipoxin A4, which has anti-inflammatory properties, levels go up during the resolution phase and remain elevated.

 

With lipoxin A4, cells that are coming in are all on their own block.  Neutrophils are infiltrating the tissue within minutes and hours, and then there’s a class switch that occurs. This was mimicked with peripheral blood through the neutrophils. However, in the pouch in the mouse, as prostaglandin E2 levels increased, they reached a threshold with peripheral blood neutrophils.  Their intracellular levels turn off leukotriene production and turn on translational regulation of the enzymes involved in lipoxin biosynthesis. Lipoxins are very potent chemoattractants, but not from neutrophil aspects. They provoke trafficking in monocytes but in a non-phlogistic fashion. Lipoxins also stimulate the uptake of apoptotic PMN by the macrophages.

Reporting the alpha signal is significant because the initial events that turn on the inflammatory response in an ideal setting signal and has governance over later events.

What is the impact of aspirin in the system? 

Aspirin dampens the early phase and is unique because it turns on specific aspirin-triggered lipid mediators. Aspirin acetylation cytologically blocks prostaglandin generation, but when COX-2 was identified, the assimilation of COX-2 does not kill the enzyme activity in vascular endothelial and mucosal epithelial cells. However, prostaglandin generation is turned off; the acetylated enzymes convert endogenous arachidonic acid to 15-R HETE that’s picked up by neutrophils.  This turns off leukotriene biosynthesis resulting in lipoxins that are epimeric at the 15 position (illustrated below as carbon 15, which is R rather than S configuration). That actually gives them a longer bio half-life in vivo because this is the site of deactivation. These mediators dampen PMN transmigration, leakage, cytokine release, etc. They also promote the uptake and removal of apoptotic cells; lipoxins, particularly epi-lipoxin A4 is anti-fibrotic.

 

Low dose aspirin initiates human ATL production

What about selective COX-2 inhibitors? Unfortunately, one of their side effects is that they block the production of endogenous aspirin-triggered lipid mediators. For example, a mouse model of leukotriene B4: If added topically, there is a neutrophilic infiltration to that tissue. It causes local leakage permeability changes that can be easily illustrated with methylene-blue dye. If an aspirin-triggered lipoxin is added topically on the mouse ear, that damage can be prevented. This is not restricted to aspirin. Recent work has shown that statins can also trigger the biosynthesis of 15-epi lipoxin A4 in cardiovascular tissue.

Human ALXR GPCR transgenic mice: diminished peritonitis

At the crystal level, serine 530 gets acetylated. This comparison illustrates how inserting molecular oxygen to make prostaglandin generation, arachidonic acid is converted to 15R-HETE so the enzyme cyclo-oxidase 2 becomes a 15R lipoxygenase. To investigate if this really happens in humans, different doses of low-dose aspirin were administered in a randomized trial. At any mix, there is a reduction in thromboxane monitored by thromboxane B2, and acetylation of COX-1 that blocks thromboxane generation is apparent. In healthy individuals there is an increase in endogenous 15-epi-lipoxin A4 generation, or aspirin-triggered lipoxin.  With age, men don’t seem to do as well as women in terms of their ability to generate aspirin-triggered lipid mediators.

Lipoxin works at multiple levels and on multiple cellular targets regulating GPCRs through phosphorylation events. The ALX receptor is also present on monocytes and it’s coupled to chemotaxis.  There’s also regulation of growth factor receptors through GPCR cross-reaction. A series of stable lipoxin and aspirin-triggered lipoxin analogs were generated to study them in more detail in vivo because glycosylators and lipid mediators are rapidly generated––they act and then they’re inactivated. In the case of lipoxins, there’s a delay in the turn-on, and once they’re made they evoke their responses, but then rapidly turn off. They’re turned off by dehydrogenation at the 15 position.

Another experimental approach is to come in from the other direction––taking the human lipoxin A4 receptor to look at an acute inflammatory response to zymosan.  In a transgenic mouse a dampening of the magnitude and a shortening of the inflammation/resolution time is apparent. This reduction in magnitude is shortened duration, and one can view this as an enhanced resolution.  This is not work that is restricted to mouse models. Research from a group in China tracked acute post-streptococcal glomerular nephritis and its natural resolution. The blood values of lipoxin A4 went up and down, urine values went up and stayed up, and leukotriene levels went down, both peripherally with infection and then with resolution in the urine. This shows that the temporal relationship does occur in humans, and there are a number of studies like this that have been published by other groups.

Using the mouse model for a more systematic approach, lipidomics and proteomics along with trafficking have been examined.  This approach has been used this with a number of model systems from skin and oral inflammation to stroke:

 

Fatty acids

A lot of clinical literature touts the benefits of omega-3s, but people don’t really pay attention to much of that literature because very high amounts appear in many of the studies. The mice in the lab had very high levels of EPA and DHA because their diet was very high in omega-3s.  Veterinarians were taking this literature to heart and enforced a diet.

AA, EPA and DHA are essential fatty acids––fatty acids that are required in the human diet, but not produced by human cells.

Resolvin E1 (RvE1)

Biosynthesis, stereochemistry and actions

In the resolving phase the first structure in the resolving exudates was identified as RvE1 for resolution phase interaction products. The structure and stereochemistry of each of the alcohol groups, and RvE1′s ability to regulate neutrophils, stop neutrophils at picogram and nanogram levels.

Stereospecific total organic synthesis

RvE1 regulates adhesion molecules; this is critical against the biological action. They also regulate IL‑12 production by dendritic cells and dendritic cell migration.

E Series Resolvin Biosynthesis: temporal 5-LO function

The receptor chemR23 is present in the human mononuclear cells in response to RvE1 and on a dose basis, RvE1 is 1,000 times more potent than aspirin. This is enzymatically converted to a 5S(6)-epoxy and then another enzyme to make RvE1, which works in a number of murine models. A dihydroxy version of this pathway called Resolvin E2 (RvE2) is also anti-inflammatory in in vivo models, but works at different sites of action than RvE1.  In the absence of aspirin, there is clearly generation of 18-EPE, thought to be in vivo in humans, and generated via P450-like mechanism.

Cell-type specific actions

Resolvin E1 acts as an agonist to chemR23 and GPCR. Radiolabels, made to study this finding, showed the first evidence for receptors that could mediate some of the actions of fish oils. It worked as a partial antagonist and agonist of BLT1 regulating neutrophil responses.

Stable analogs in vivo

As in the case of most autocoids, RvE1 is generated in response to stimuli, act locally, then rapidly metabolized via enzymatic inactivation.  For example, the major metabolic route for RvE1 in mouse lungs is the conversion to 18-oxo-RvE1, which is inactive in regulation PMN infiltration in vivo.  Therefore, stable analogs of RvE1 were designed and synthesized, including RvE1 methyl ester, and 19-parafluoro-phenoxy-RvE1.  These stable analogs resist enzymatic inactivation and prolong bioavailability and bioaction in vivo, thus are useful tools to investigate RvE1’s action in vivo.

Using the stable analogs, RvE1’s protective action was further determined in complex disease models, and are active in nanogram to low microgram quantities, via different routes of administration, such as IV, IP and topical.

Human localized aggressive periodontitis: LAP 

RvE1 is inactivated through dehydrogenation. Mapping this out enables the creation of stable analogs of Resolvins, which prevent this inactivation. Studies of RvE1, as well as the analogs in a number of systems, show that they are IV active, IT topical and very low doses.

Using periodontal disease to examine the role of the neutrophil in periodontal pockets requires controlling neutrophil infiltration. Neutrophils are solely responsible for periodontal ligament destruction and looseness of the tooth, which can lead to tooth loss.

RvE1 protects from inflammatory bone loss

Using a rabbit model, the entire ligature alone around the tooth emulates the nidus for a plaque formation. If this is doused with the clinical isolate of Porphyromonas gingivalis, a very vigorous neutrophilic infiltration is caused. This can also be thought of as a reperfusion injury, and it can be monitored radiologically.  The figure below shows a very prominent loss of bone, within a few weeks.

Therapeutic dosing of RvE1 protects bone and cartilage destruction in rat CIA model. RvE1 actually stimulates new bone growth in animal models, and we hope to see that in the future studies with humans.  This finding could actually translate. Resolvyx, a startup company, has looked at RvE1 and found it protects bone and cartilage degradation both in mouse models of RA and classic collagen-induced arthritis models.

Fat-1 transgenic mice rich in endogenous omega-3

The fat-1 transgenic mouse has the enzyme responsible for elongation of fatty acids. This enzyme eliminates the diet control completely, and provides for tissue levels, EPA and DHA without having to add this into the dye. Once profiled, resolvins and protectins are made, and the fat-1 mouse is protected.

RvE1 protects against TNBS-induced colitis

When replicated, this experiment with TNBS colitis and RvE1 shows reduction in lymphocytic infiltration by histology in the fat-1 transgenic mouse.  NPL is a marker of neutrophils, and it is also reduced. Some antibody production is reduced, and certainly all the inflammatory markers, INOF, COX, TNF-alpha are brought down, but most importantly, the survival level of these mice changed dramatically, and their lives were prolonged.

RvE1 dampens airway inflammation

In an animal model of allergic asthma, RvE1 dampens airway inflammation. Mice were sensitized and aerosol challenged with ovalbumin. Shown below in blue, mice that received RvE1 at 100ng iv together with each aerosol challenge, showed marked decrease of eosinophils, macrophages and lymphocytes in broncho-alveolar lavage. When RvE1 was given after aerosol challenge, shown in red, it retained the ability to reduce the leukocyte umbers in broncho-alveolar lavage. These results emphasize RvE1’s capacity to accelerate resolution of allergic airway inflammation.

 

Interestingly, RvE1 treatment led to significant lower amounts of pro-inflammatory LTB4 and higher amounts of LXA4 in the lungs.  In addition, RvE1 reduced the production of IL-6, IL-17 and IL-23.  These results established RvE1’s potent action in accelerating resolution of airway inflammation, and identified novel components in RvE1’s mechanisms of action, including eicosanoids and Th17 cytokines.

Resolvins & protectins: new mediator families

There’s a lot of literature on how DHA is sequestered and is neuroprotective in the neural tissues of the brain. The air pouch had C22 structures that remained–Resolvin D1, 2, 3 and 4 that were very important in dampening neutrophilic infiltration. Tracing back also finds that DHA is a precursor and can transform to dihydroxy compounds, which are called protectins, one of which is neuroprotectin D1. The biosynthesis showed initially that DHA in the presence of aspirin and COX-2 was converted to 17R HPED. In the absence of aspirin, the lipoxygenase carries this out to make these resolvins, which are rather potent.

How potent are they?  Based on 100 ng in equal dosing, the resolvins IV (in murine peritonitis), the aspirin trigger and the endogenous S-pathway are essentially 50% reduction in neutrophilic infiltration, and IV RvE1 is about 75% reduction. They are very, very potent. The key to this is the total organic synthesis because there are very small quantities that are made in the mouse models, and RvE1, D1 and protectin D1 are synthesized in the lab. This also gives the ability to assign stereochemistry to confirm the original structural elucidations from the murine air pouches and in vivo inflammation models as well as provide the tools to go back and scale up and ask more questions about their mechanisms of action in vivo.

General strategy: total organic synthesis resolvins and protectins
An introduction to the study of experimental medicine

Looking systematically at the exudate and applying this approach of being quantitative, a number of indices are introduced to look at neutrophilic infiltration and then pinpoint the site of action of these compounds when added back. The resolution interval is just the interval where 50% TMN is dropped and get the non-phlogistic regrouping of monocytes to the tissue. With neuroprotectin D1 and each of the resolvins it can be demonstrated that the resolution time can be shortened.

Mechanistically, it can be added at time zero and dampen the amplitude with the aspirin-triggered lipoxins or RvE1. The DHA-derived compounds can be added at this point, but they can also be added at the maximum neutrophilic infiltration point and then expedite the loss of the neutrophil from the site and then return to homeostasis, at least in murine models. This opens up resolution pharmacology to be able to show for the first time that endogenous resolution mechanisms can be stimulated.

The cytokines, or RvE1 or PD1, for lipoxin A4 all lead to a reduction of chemokines and proinflammatory cytokines and generally they all lead to an increase in IL-10, but they each have very different fingerprint and site of action if we are regulating cytokines.

One of the key points is how important the macrophage phagocytosis is here; lipoxin, Resolvin and PD1 stimulate the uptake of apoptotic PMN. This is a clearing out of the dying cells at the exudative site.

Pro-resolving mediators potent agonists of macrophage phagocytosis

The right figure shows that this is not only apoptotic PMN but enhanced phagocytosis of microbes as well.  These compounds’ novel aspect of their action as a genus is that they dampen neutrophilic infiltration, but they enhance the clearance of apoptotic cells.  They also have an antimicrobial action by stimulating endogenous defensive mechanisms from epithelial cells. Using those indices, for example, inhibitors like COX-2 inhibitors actually prevent resolution completely.

What about the substrates and their availability in humans? There’s very little data in the literature about EPA, DHA and the actual levels in humans.

Deuterium label can be added to track EPA and DHA by mass spectrometry. This increases the molecular weight by 5 units to easily discern it in inflammatory exudate. If this IV is injected in a mouse, how rapidly does this turn up at the site of inflammation?

EPA and DHA are rapidly apparent in non-esterified form, meaning that it can be converted to resolvins. There seem to be two bursts: one initially and then one in the resolution phase. Without inflammation there’s virtually no EPA or DHA identifiable within the exudate.  The leukocyte count and protein edema levels tell us that without edema, EPA and DHA can’t be seen entering into the exudate. Therefore, edema formation is also playing a very important role in regulating the outcome of resolution. EPA and DHA in this setting are most likely being carried by albumin and other serum proteins, it does not need to be esterified.

Novel microfluidics chamber & single cell with 1 drop of blood <5min
This is a chamber that was designed by Nematoner and his colleagues. It usually takes two to three hours to isolate peripheral blood neutrophils and the nice, isolated cells to work with from peripheral blood.  The advantage of this system is it can be done in less than five minutes.

The anatomy of the chamber:

Cell movement can be seen and basically there’s a tethered P selectin on the surface with some chemical modifications imposed. Done with resolvin D1 present, and then calculating the number of cells, the cells move along and they have this bipolar sort of chemotaxin morphology. As soon as they see resolvin D1 they round up, stop and no longer chemotax. About five minutes later they are able to chemotax again and change shape. It’s a very rapid change of shape, and this is important in the action.

Functional decoding metabolomics
Is it the precursor or the product that’s needed? The cells track along before admission of either RvD1 or DHA at equal and lower amounts. The cells stop, whereas DHA at equal and lower amounts move just like control. This gives a nice opportunity to assay very rare compounds that are generated within the inflammatory milieu and determine if they have biological action.

Reperfusion associated remote organ injury: a murine model

The figure shows the second organ injury reperfusion injury that emulates what one might see in surgery if blocked with clamps. When the reflow starts, the leukocytes get consumed on primarily neutrophils on the hind limbs. They hit the lung, and leave it damaged. Reperfusion injury is very important in the joint as well because as inflammation moves along and pressure is applied, it causes recurrent hypoxia and reoxygenation and a milieu for an acute injury.

In this model, if given resolvin D1 versus DHA, DHA is not active, RvD1 is and RvE1 is not.  In this system, all the concepts come together and show that this lung injury can be reduced with RvD1, as well as make more stable analogs that are easier to handle. Most importantly, RvE1, which was not active in the system, can be used to introduce functional moieties to increase its availability and half-life.

Conclusion
EPA and DHA circulating levels are rapidly transformed at sites of inflammation to resolvins and protectins. This is part of resolution to homeostasis, protecting in second organ injuries, and can be thought about as joint destruction or uncontrolled inflammation such as periodontal disease or arthritis.

Resolvins and lipoxins each bind specific GPCR and cellular targets. They have specific GPR, CR and cell targets. They are very potent in animal models, and that’s been extended by a number of groups recently. Therapeutically, all the current available pharmacopeia are directed towards inhibition. The new concept that drops out of this work is to stimulate resolution with agonists.

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Updated: July 10, 2012

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