Neutrophil Extracellular Traps as Therapeutic Targets for Inflammatory Disease
Rory R. Koenen
DOI : 10.3844/ajptsp.2014.200.202
American Journal of Pharmacology and Toxicology
Volume 9, Issue 4
Neutrophilic granulocytes, orneutrophils are the most abundant circulating white blood cell and constitute afirst line of defense against pathogens. They are characterized by a largepolymorphic lobular nucleus and the presence of many secretory granules intheir cytoplasm. Neutrophils are essential for a functional host defense, as isevidenced by the occurrence of severe infections in individuals with decreasedneutrophilnumbers or neutrophil function. Neutrophils bind to and subsequentlyphagocytize bacteria, which are effectively destroyed by reactive oxygenspecies and digestive enzymes in the neutrophil's interior. Being secretorycells, neutrophils also release numerous factors with antimicrobial orimmunomodulatory activity.
A decade ago, afurther mode of the neutrophil's antimicrobial action was discovered, therelease of its nuclear DNA content to the extracellular space. The resultingextracellular DNA exists in complex with histones and with a variety ofneutrophil effector proteins, e.g., Neutrophil Elastase (NE), Myeloperoxidase (MPO),LL37 and proteinase 3. This structure was named "Neutrophil ExtracellularTrap (NET)" and the process of NET-release was termed "NETosis".A variety of pathogenic bacteria can induce the release of NETs fromneutrophils and the predominant opinion is that NETs function to entrapbacteria, which are immobilized and subsequently killed by the antimicrobialfactors that populate the NET.
Interestingly, the potential to induceNET release in neutrophils is not restricted to pathogens. For example,activated platelets have also been shown to induce NETosis when bound to neutrophils.This finding has important implications for our understanding of (auto-) immune-relateddisorders, as this would mean that NET formation can also be detrimental to thehost organism. Indeed since their discovery, an adverse action of NET releasehas been implicated in many auto-immune and inflammatory diseases e.g.,vasculitis, lupus, rheumatoid arthritis and atherosclerosis, but also in acutelung injury, venous thrombosis and myocardialischemia/reperfusion injury. In these disorders, NETs may bothconstitute a source of auto-antigens as well as being a propagator ofinflammation. Thus, counteracting the formation of NETs, or their effects,would be an attractive approach for the prevention or treatment of thesediseases. Of note, circulating NETs including their remnants and constituentsrepresent interesting biomarkers for NET-driven disorders. A semi-quantitativemethod for the quantification of NETs in body fluids of humans and mice isbased on the association of DNA with Myeloperoxidase (MPO), an enzymeresponsible for hypochlorite production by neutrophils. Using specificantibodies, DNA is captured from the specimen and MPO activity is subsequentlymeasured using specific substrates, or vice versa. However at present,the semi-quantitative nature of this method only allows the measurement ofrelative changes of NET content among samples.
A number ofrecent studies focused on the potential usefulness of NET-targeted approachesfor the treatment of a number of disorders. One of these approaches is theinhibition of Peptidylargininedeiminase 4 (PAD4), a histone-modifying enzyme that catalyzes the conversion of arginine tocitrulline that was identified to play an important role in NET release byneutrophils. Mice with a genetic deletion of PAD4 showed impaired hostdefense during a model of bacterial necrotizing fasciitis,which was explainedby the inability of PAD4–/– neutrophils to release the NETs neededto inactivate the pathogen. In addition, the development of venous thrombosis,which was recently described to be a NET-driven pathologic event, wassignificantly reduced in PAD4–/– mice. A mouse model of lupus wasshown to depend on the formation of NETs by neutrophils, as neutrophils derivedfrom diseased mice showed increased NET formation and sera from diseased micecould induce NETosis in healthy control mice. Mice with lupus also showedmeasurable circulating antibodies directed against neutrophil intracellularcomponents. In this and other studies, potential of PAD as a therapeutic targetwas highlighted by a recently described synthetic inhibitor, termed Cl-amidine, which irreversibly inactivatesPAD by covalently binding to a cysteine residue in the active site. Treatmentof neutrophils with Cl-amidine resulted in decreased histone citrullinationandreduced release of NETs from neutrophils. In addition, administration ofCl-amidine to mice with lupus reduced the deposition of MPO-containing immunecomplexes in the kidney and ameliorated endothelial dysfunction. Although thesefindings indicated that pharmacologic inhibition of PAD (and thus NETformation) might be an attractive target for the treatment of lupus, the studycontained no data about the clinical severity of the lupus in mice without orwith Cl-amidine treatment.
The potentialbenefit of a pharmacologic inhibition of PAD was also investigated in a mousemodel of atherosclerosis. An increase of NET formation was observed in the seraof hyperlipidemic ApoE–/– mice during the administration of ahigh-fat diet and the sera of atherosclerotic mice were able to induce NETosisin isolated neutrophils. Advanced atherosclerotic lesions (after 11 weeks ofdiet) showed increased histologic staining of citrullinated histone 3, comparedto those of 8 week old ApoE–/– mice. Repeated injection ofCl-amidine reduced atherosclerotic lesion formation in the aortic trees of ApoE–/–mice after 11 weeks of high-fat diet and decreased both neutrophil andmacrophage content in the lesions. Interestingly, treatment withCl-amidineappeared to be ineffective after depleting neutrophils in the ApoE–/–mice, indicating that the primary target of Cl-amidine was indeed theNET-forming neutrophil. However, it must be mentioned that neutrophil-depletionalone almost completely abrogated atherosclerotic lesion formation in thoseApoE–/– mice, which would leave little room for a further preventionof atherosclerosis by Cl-amidine.
A differentapproach for preventing NET formation was investigated by Rossaint in a mousemodel of Ventilator-Induced acute Lung Injury (VILI). Given that activatedplatelets can induce NET-formation, the authors investigated the effect ofplatelet-depletion on the extent of VILI in mice. Depletion of plateletsresulted in improved clinical parameters and less circulatingplatelet-neutrophil complexes after VILI in mice. Moreover, a decreased contentof NETs was observed in lung sections of platelet-depleted mice after VILI,compared with non-depleted mice. Concomitant integrin-mediated contact with theextracellular matrix of adhesion molecules along with physical binding toactivated platelets was found to be necessary to induce NET release byneutrophils. In addition, the chemokines CCL5 and CXCL4 were identified asplatelet-derived soluble factors required to stimulate the release of NETs.However, neither CCL5 nor CXCL4 alone was able to induce NET-formation, onlythe combination of CCL5 and CXCL4 triggered NETosis. Previous studies havedemonstrated that these chemokines exist as a particularly potent heteromericcomplex and a peptide, termed MKEY, that disrupts the CCL5-CXCL4 complex wasshown to reduce atherosclerosis in ApoE–/– mice. Interestingly, MKEYwas also able to prevent NET-release by platelets. Administration of MKEY inmice during the course of VILI reduced the number of circulatingplatelet-neutrophil complexes, decreased alveolar neutrophil accumulation andprevented NET-formation, at least when applied early after the onset of VILI. The above findings are in agreement withthose from previous studies, where platelet depletion or pharmacologic plateletinhibition protected mice from experimental lung injury. Thus, also functionalinhibition of platelet-secreted chemokines might prevent NET formation andmight be a beneficial therapeutic approach for the treatment of auto-immune orinflammatory diseases.
Taken together,NETs appear to be an attractive therapeutic target, as they are implicated inthe pathophysiology of a variety of inflammatory disorders. However, most ofthe observations were done using experimental models in mice, whose neutrophilcounts and physiology differ from those in humans. In addition, a recent studypresented a patient with an almost complete deficiency in several neutrophilgranule proteins (e.g., NE, LL37 and protease3) and with impaired NET-formation, yet who suffered only from mild clinicalimmunodeficiency. Thus, appropriate methods for quantitativeNET-detection and clinical studies in humans are required to establish thepotential benefits of preventing NET-release in neutrophils.
Conflict of Interest Statement
The author is ashareholder of Carolus Therapeutics Inc., San Diego, CA.
Sources of Funding
This work was supported byDeutsche Forschungsgemeinschaft (DFG Ko2948/1-2, SFB 1123 and FOR809) and theNetherlands Foundation for Scientific Research (ZonMW VIDI 016.126.358) awardedto R.R.K.
© 2014 Rory R. Koenen. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.