An Insight Into the Pro

8/6/2019 An Insight Into the Pro

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An insight into lipid mediators and exploring the pro-

resolving action of RvD1

ABSTRACT

Inflammation is not only the effect of a pathogenic insult to the organ or tissue but it has also been

linked with diseases such as cancer, asthma and Alzheimers. The resolution of inflammation

involves removal of the leukocytes and pathogenic debris from the site, which is initiated by pro

resolution lipid mediators like resolvins and lipoxins. Characterization of the biochemical pathways

leading to resolution might lead to the identification of novel targets that can be exploited for

innovative anti-inflammatory drug discovery. The paper provides insights into the pro resolving

actions of Resolvin D1(RvD1). It is shown that RvD1 specifically binds to human phagocytes and

improves the phagocytic clearance functions of human M. Specific RvD1 recognition sites on

human leukocytes was obtained by synthesizing [3T]-RvD1. RvD1 also enhanced macrophage

phagocytosis of zymosan and apoptotic PMNs, which increased with over-expression of human ALX

and GPR32 indicating that RvD1 specifically interacts with both ALX and GPR32 on phagocytes to

achieve its role in resolution of inflammation.

M ain paper

Inflammation is widely appreciated in the pathogenesis of many human diseases. These

extend from the well-known inflammatory diseases such as arthritis and periodontal disease

to those not previously linked to aberrant inflammation that today include diseases affecting

many individuals such as cancer, cardiovascular diseases, asthma, and Alzheimer¶s

disease. Challenge of host tissues by microorganisms, tissue injury or surgical trauma leads

to the release of exogenous and endogenous chemical mediators that in turn give rise to the



initial cardinal signs of inflammation, rubor (redness), calor (heat), tumor (swelling) and dolor

(pain).

The exogenous mediators include m icrobial peptides that act as chemo-attractants to

recruit neutrophils to the site of challenge, where they phagocytose invading microorganisms

and cellular debris. Within the neutrophils, newly formed phagosomes mature to form

phagolysosomes by fusing with lysosomal granules, which contain degradative enzymes and

produce reactive oxygen species (ROS) to kill trapped microorganisms or degrade cellular

debris. The initial inflammatory response functions to protect the host and, ideally, its timely

resolution ensure that it is self-limiting.

Acute inflammation has several programmed fates including progression to chronic tissue

fibrosis and the ideal outcome of complete resolution. Once the initiating noxious materials

are removed, the inflammatory reacti on must be resolved to prevent the inflammation from

spreading, becoming chronic or causing disease. Resolution of inflammation, or its

catabasis, is the reduction or removal of leukocytes and debris from inflamed sites, enabling

the return to homeostasis. The resolution process is rapidly initiated after acute challenges

by cellular pathways that actively biosynthesize local, specialized, dual -acting anti-

inflammatory and pro-resolution lipid mediators . Prostaglandin E2 and prostaglandin D2, for

example, stimulate the switching of arachidonic -acid-derived lipids from leukotriene B4

production to lipoxin A4 production and then the switching of lipid mediator families to

produce anti -inflammatory and pro-resolution lipid mediators, such as Eseries and Dseries

resolvins and protectins. Alternatively, chronic inflammation can result from excessive and/or

unresolved inflammatory responses and can lead to chronic disorders.

The resolution of inflammation is now considered to be a distinct process from anti-

inflammatory processes. Specific lipoxins and members of the resolvin and protectin families

provide potent signals that selectively stop neutrophil and eosinophil infiltration; stimulate

non-phlogistic recruitment of monocytes and activate macrophage phagocytosis of

microorganisms and apoptotic cells .



Resolvins

The first resolvin was identified in exudates collected from inflamed murine dorsal air

pouches in the spontaneous resolution phase and was so -named because it proved to be a

potent regulator of resolution.

There are several steps in the initial inflammatory cascade triggered by cytokines.

Inflammatory products of arachidonic acid oxidation (omega -6) including inflammatory

prostaglandins (PGE2) and lipoxins (LTB4) are released from infiltrating my eloid cells. In

contrast, products of eicosapentanoic acid (omega -3) oxidation, PGE3, and LTB5, have anti -

inflammatory activities. Products of omega-3 fatty acid oxidation include resolvins of the E

series (RvE1 and RvE2), which are found naturally in nearly all inflammatory sites in

mammals. The D series of resolvins are derived from docosahexaenoic acid (DHA). In

general, resolvins are part of the anti -inflammatory portfolio that coexists with inflammation.

Synthetic forms of RvE1 are currently in clinical trials for treating ocular diseases and other

local inflammatory conditions. In animal models of sterile inflammation, RvE1 suppresses the

number of infiltrating neutrophils and macrophages as well as decreasing expression of the

genes encoding TNF-a, IL-1b, and VEGF. The anti-inflammatory properties of omega -3 fatty



acids include suppression of IL-1b and TNF-a production, and the mechanism of action of

omega-3 fatty acids may include boosting production of resolvins of the D and E series .

Resolvins control inflammation at many levels, by reducing peritonitis and skin inflammation

,protecting organs from reperfusion injury and neovascularisation.

Receptor s and actions of RvD1:

Ov er v iew

The authors demonstrate that RvD1 actions on human polymorphonuclear leukocytes

(PMNs) are pertussis toxin sensitive, decrease actin polymerization, and block LTB4 -

regulated adhesion molecules (2 integrins). Specific RvD1 recognition sites on human

leukocytes was obtained by synthesizing [T] -RvD1. RvD1 also enhanced macrophage

phagocytosis of zymosan and apoptotic PMNs, which increased with over-expression of

human ALX and GPR32 and decreased with knockdown of these G-protein-coupled

receptors. Also, ALX and GPR32 surface expression in human monocytes was up-regulated



by zymosan and granulocyte-monocyte±colony-stimulating factor. These results indicated

that RvD1 specifically interacts with both ALX and GPR32 on phagocytes and suggest that

both of them play a role in resolving acute inflammation.

Resolvin D1 (RvD1) biosynthesis and structure were established and its stereochemistry

assigned 7S, 8R, 17S-trihydroxy- 4Z, 9E, 11E, 13Z, 15E, and 19Z -docosahexaenoic acid.

RvD1 was earlier found to limit PMN infiltration at nanogram levels in murine peritonitis and

block trans-endothelial migration of human PMN ¶s as well as enhances phagocytosis by

human macrophages (M). RvD1 was also found to directly act at a single -cell level in micro

fluidic chambers to stop human PMN migration to interleukin -8.

Experiments and results

Resolvins control inflammation at many levels, by reducing peritonitis and skin inflammation,

protecting organs from reperfusion injury and neovascularisation. Human PMNs incubated

with RvD1 (10 nM) resulted in a decrease in actin polymerizatio n. To assess whether

GPCRs are involved in RvD1 signal transduction, PMNs were treated with either pertussis

toxin (PTX) or activated cholera toxin and then incubated with RvD1. Interestingly, RvD1 did

not stimulate intracellular Ca 2+ mobilization or activate cAMP with human PMNs, suggesting

that these classic second messengers are not activated by RvD1 signalling in these cells.

Nuclear receptors may evoke anti-inflammatory responses. In the system designed by the

authors ,where HEK-293 cells were cotransfected with constructs ofligand -binding domains

RvD1 did not directly activate mouse peroxisome proliferator-activated receptor (PPAR)-, -

, and - or human retinoid X receptor -. These results indicated that RvD1in its anti -

inflammatory dose range did not activate these specific nuclear receptors.

The next aim was to determine whether leukocytes have special binding sites for RvD1.To

achieve this tritium labelled RvD1 was prepared. Competition binding was performed with

RvD1, LXA4, and the annexin 1 -derived peptide Ac2 -12, which is an anti-inflammatory

peptide ligand that binds to ALX. The homo-ligand RvD1 displaced [3H]-RvD1 binding to

cells, and the extent of displacement was taken as 100% specific binding.



of these ligands with ALX. Again, using compound 43 with ALX stable -arrestin cells gave a

dose dependent activation with an EC50 of 2.1 × 10 -12 M . Notably, compound 43 was

unable to activate the -arrestin system. These results suggest that RvD1 interacts with both

ALX and GPR32.

Expression of GPR32 and ALX in Human M Regulates RvD1-

Stimulated Phagocytosis.

Because complete resolution of acute inflammation involves phagocytosis and clearance of

apoptotic PMNs by M from the inflammatory milieu, which is enhanced by resolvins, we

investigated whether RvD1 can enhance the phagocytic activity of human M. Human M

exposed to RvD1 following differentiation of peripheral blood monocytes enhanced their

ability to ingest zymosan and apoptotic human PMN¶s in a dose-dependent manner. RvD1

showed an increase in phagocytosis of zymosan particles in transfected M that was further

increased in M over expressing either GPR32 or ALX. Conversely, M with transient small

hairpin RNA (shRNA) knockdown of ALX or GPR32 showed a decrease in RvD1 -stimulated

phagocytosis response.

Conclusions

Resolution of inflammation is an active process with many control points, regulated by a

unique genus of chemical mediators that are anti-inflammatory and proresolving. Among

these, RvD1 possesses potent actions demonstrable at the single -cell level with leukocytes

in a microfluidics chamber.



[3H]-RvD1 prepared by total organic synthesis was used to identify high-affinity cell-surface

recognition sites for RvD1 on human leukocytes, giving a Kd of 0.2 nM , which is within the

range of its levels measured in murine cells and tissues, i.e., >75±300. Of interest is the fact

that LXA4 partially displaced [3H]-RvD1 specific binding to human PMNs. A screening

system for identifying receptor candidates, which tests the ability of receptor±ligand coupling

to counteract TNF- -stimulated NF-B activation, gave candidate GPCRs, namely ALX, a

LXA4 receptor, and an orphan, GPR32. Interestingly, GPR32 and ALX surface expressions

in human monocytes were up -regulated on exposure to GM-CSF and zymosan but not by

exposure to TNF- or TGF-. This correlates with RvD1 actions on macrophages that

enhance clearance of zymosan particles.

RvD1 interactions with human ALX and GPR32 were further evaluated using another

reporter system, one that monitors, in a ligand-dependent fashion, coupling of intracellular -

arrestin with GPCR cytoplasmic domains. This system permits monitori ng of ligand±receptor

interactions without classic second messengers involved. This was needed because RvD1

did not directly evoke Ca2+ mobilization nor change cAMP levels. This system employed

recombinant cells engineered to stably over-express ALX, ChemR23, or GPR32, each

tagged with the Pro-Link peptide of -gal and -arrestin linked to the enzyme acceptor (EA)

fragment of -gal. With this system, LXA4 was also identified as interacting with GPR32,

suggesting that this GPCR may be part of a cluster of receptors transducing proresolution

signals with lipid mediators. Along these lines, an ALX agonist ² denoted compound 43 and

recently identified via a medicinal chemistry screen with anti-inflammatory actions in vivo ²

was also found to activate both ALX and GPR32 in stable -arrestin cell systems. The -

arrestin signal in this system activated by agonists showed a more than twofold increase

compared to the vehicle . Transient transfection of human M with expression vector for

either ALX or GPR32 increased the ability of RvD1 to enhance phagocytosis, suggesting

that the RvD1 response is both ALX - and GPR32-dependent. These findings functionally

validate results from the reporter systems used to identify ALX and GPR32 as interacting

with RvD1.



LXA4 and RvD1 share some anti-inflammatory and proresolving actions in human and

murine systems , yet each is biosynthesized at different time intervals during resolution

and via distinct biosynthetic routes . In summary, RvD1 specifically binds to human

phagocytes and activates two separate GPCRs, namely ALX and GPR32. RvD1 enhances

the phagocytic and clearance functions of human M, which were increased by these

GPCRs. Taken together, these findings suggest that RvD1 executes its proresolving actions

within human tissues via its interactions with ALX and GPR32.

Further scope

The recently uncovered families of EPA - and DHA derived chemical mediators reviewed

here, namely the resolvins and protectins, now open new possibilities to design µresolution -

targeted¶ therapies for controlling unwanted side effects of excessive inflammatory

responses. These omega-3-derived lipid mediators are autacoids and function as agonists at

temporally and spatially distinct steps in the progression of inflammation and its resolution.

As many current and widely used drugs were developed without knowledge of their impact in

resolution processes, some agents, such as selective COX2 inhibitors and certain

lipoxygenase inhibitors, have now been shown to impair the tissue programmes of

resolution, thereby delaying the return to homeostasis, whereas others, such as

glucocorticoids, aspirin, cyclin -dependent kinase inhibitors and statins, seem to work in

concert with endogenous pro -resolution processes. COX inhibitors reduce the amplitude of

and the cardinal signs of inflammation by inhibiting prostanoid biosynthesis.



Therefore, combining pro-resolution molecules with lipoxygenase or COX pathway

antagonists may be a useful strategy to restore resolution and control excessive

inflammation. Neverthel ess, before resolution pathways can be harnessed for the treatment

of human diseases, several important questions need to be addressed: will it be possible to

treat organ-specific inflammatory disorders with different pro-resolution mediators and/or

their analogue mimetics in a tissue -specific manner? Will dietary supplementation with

intermediates and/or precursors be beneficial for the local production of pro-resolution

mediators? Will knowledge of these new pathways lead to an increased appreciation of

potential deleterious effects of excessive DHA and EPA intake that could lead to increases in

auto-oxidation of these fatty acids that can damage cells and tissues? Finally, given that

lipoxins, resolvin E1 and protectins act on T cells, dendritic cells and phagocytic cells, might

nutrition represent a molecular link between the innate and adaptive immune systems?

The results of this paper provide valuable insights into the receptors for Rvd1, and

comparison with that of RvE1 shows important similarities as well as differences. These

results may provide insights into the actions of other lipid mediators like neuroprotectins. A

singular venue leading from this research is insights into the very cause and resolution of



inflammation, an aspect which if comprehended more would lead to breakthrough drugs. A

range of therapies exist for the treatment of inflammation -driven diseases such as asthma,

rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, Crohn's disease and

systemic vasculitis. However, such therapeutics has undesirable side effects. For instance,

steroids can cause osteoporosis and impair wound healing, whereas novel selective

inducible cyclooxygenase inhibitors might reduce protective vascular prostacyclin synthesis,

leading to an increased risk of thrombosis. Indeed, experience with tumour necrosis factor -

(TNF- )-neutralizing therapy has also revealed several complications. Clinical studies

indicate that TNF- -neutralizing therapy should not be given to patients with cardiac failure

or a history of demyelinating disease. In addition, increased infection rates can occur,

especially the activation of latent tuberculosis, although other opportunistic infections su ch

as listeriosis, pneumonia and aspergillosis have also been reported. These unwanted side

effects stem from the inhibition of essential endogenous factors that have a role in normal

physiology or, in the case of anti -TNF- , by dampening down the host's a bility to adequately

deal with infection, given that the innate inflammatory response is a beneficial defensive

event. Detailed characterization of the biochemical pathways leading to resolution might lead

to the identification of novel targets that can be exploited for innovative anti -inflammatory

drug discovery. Novel compounds based on the mechanism of action of a given pro -

resolving mediator will be modulatory in their action and likely to produce a lower burden of

side effects. There are no drugs in the clinic that are purposefully based on the elicitation of

pro-resolving pathways, with the exception of those drugs that target apoptosis. There are,

however, drugs in current usage that mediate their effects, at least in part, by triggering the

synthesis of endogenous anti -inflammatory mediators in a manner that might be related to

pro-resolution. These include methotrexate, sulphasalazine and FK506. They provide

support for the concept of activating pro -resolution pathways to treat chronic inflammation.

Further investigation into the mediators and mechanisms that are central to resolution will

bring us into a new era of inflammation research, which, if approached with creativity and



persistence, might provide numerous benefits for those suffering from inf lammation-

mediated diseases.

Referenc es

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Petasisc, and Charles N. Serhan(2010).16601665 PNAS January 26, vol. 107 no. 4.(primary paper)

2. Nathan, C. (2002) Points of control in inflammation. Nature 420, 846852 (primary review)

3. Amiram Ariel 1 ,3 and Charles N. Serhan(2007) TRENDS in Immunology Vol.28 No.4,176-183

4. Weissmann, G., Smolen, J. E. & Korchak, H. M. (1980) N. Engl. J. Med 303, 2734

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An Insight Into the Pro

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