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hyperthyroid state may be modulated depend-ing on whether the individual has brown adipose tissue. The new findings of this study may, therefore, explain the increased metabolism in hyperthyroid individuals and why it manifests differently depending on the person.

Whereas the new findings may not lead to any direct alterations in the clinical treatment of hyperthyroidism, they profoundly alter general concepts in metabolism. The study also points

to the possibilities of modifying metabolic rates through manipulations of the hypothalamus, opening new avenues for altering metabolic efficiency by activating brown adipose tissue to prevent or ameliorate obesity.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

1. Silva, J.E. Physiol. Rev. 86, 435–464 (2006).2. Lopez, M. et al. Nat. Med. 16, 1001–1008 (2010).

3. Fliers, E. et al. Trends Endocrinol. Metab. 21, 230–236 (2010).

4. Szekely, M. Acta Physiol. Hung. 37, 51–56 (1970).5. Cannon, B. et al. Ann. N.Y. Acad. sci. 856, 171–187

(1998).6. King, B.M. Physiol. Behav. 87, 221–244 (2006).7. Cannon, B. & Nedergaard, J. Physiol. Rev. 84, 277–359

(2004).8. Enerbäck, S. et al. Nature 387, 90–94 (1997).9. Cannon, B. & Nedergaard, J. int. J. Obes. (in the press).10. Nedergaard, J. et al. Am. J. Physiol. 293, E444–E452

(2007).11. Saito, M. et al. Diabetes 58, 1526–1531 (2009).12. Zingaretti, M.C. et al. FAsEB J. 23, 3113–3120 (2009).

A clear understanding of why there is so much DNA and debris in the airway is still elusive. Apoptosis of neutrophils followed by macrophage clearance is normally a very efficient process and should prevent excess debris and DNA. We also do not know why bacteria thrive in this environment. More important, why can’t we eliminate this viscous cycle of infection, inflammation and destruction and clogging of the airway in people with cystic fibrosis?

In this issue of Nature Medicine, Marcos et al.2 provide new insights into the genesis of the thick DNA-rich sputum and its implication in the pathology of cystic fibrosis. The authors show that NETs, which are the last effort of the neutro-phil to trap pathogens, accumulate in the airways, worsening lung function in indivi duals with cystic fibrosis. Moreover, activation of the neutro-phil G protein–coupled membrane chemokine receptor CXCR2 led to the formation of the NETs, and its blockage resulted in improved lung func-tion in a mouse model of cystic fibrosis (Fig. 1). This study provides new hints on how to alleviate airflow obstruction in the clinic.

Neutrophils, which are immune cells armed with defense weapons in the bone marrow, are deployed into the bloodstream. In cystic fibro-sis, numerous neutrophils invade the airway, a field of unresolved conflict, representing an unusual instance of perpetual and acute inflam-mation3. Activated neutrophils phagocytose bacteria, delivering them to their lysosomal machinery, which uses reactive oxygen species and degrading proteinases for bacterial killing. Before the intracellular killing machinery winds down and the neutrophil prepares for death, it has one more trick up its sleeve—‘NETosis’.

Cystic fibrosis is one of the most common inherited disorders in populations of European descent, occurring in about one in 3,000 births1. Autosomal recessive mutations in the cystic fibrotic transmembrane conductance regulator (CFTR) cause changes in epithelial cell Cl– secretion and Na+ absorption, result-ing in a variety of abnormalities including abnormal sweating, pancreatic insufficiency, intestinal dysfunction and thick, tenacious mucus in the airways that is difficult to clear. In fact, individuals with cystic fibrosis struggle for hours each day, using mechanical vests, inhaled medications, chest percussion and other procedures to breathe more easily.

In cystic fibrosis, the entire airway, com-posed of the trachea, bronchi and bronchioles, is draped with abnormal and adherent mucus, making it difficult to clear by the ‘mucociliary elevator’—the first line of innate host defense. This is also an attractive breeding ground for infectious bacteria, particularly Pseudomonas aeruginosa. Neutrophils, the major acute innate specialized phagocyte, attack the bacteria, but also they die trying to clear the infection. This leaves the airway littered with matrix- destructive proteinases—enzymes used by neutrophils to degrade pathogens—and DNA from neutrophils and other dead cells, which, in turn, makes the mucus even thicker, prone to limiting airflow into the lungs and causing breathlessness.

A. Murat Kaynar is in the Department of Critical

Care Medicine and Steven D. Shapiro is in the

Department of Medicine, University of Pittsburgh

School of Medicine, Pittsburgh, Pennsylvania, USA.

e-mail: sds33@pitt.edu

NET loss of air in cystic fibrosisA Murat Kaynar & Steven D Shapiro

Thick, adherent mucus in the airway causes respiratory failure—the leading cause of death in cystic fibrosis. A new study now shows how the formation of neutrophil extracellular traps (NETs) in the airway, in an attempt to kill colonizing bacteria, results in chronic cell carnage that thickens the sputum, worsening lung function in individuals with cystic fibrosis (pages 1018–1023).

NET formation, or NETosis4,5, begins in the nucleus, where chromatin mixes with histones, alkaline proteins that normally act as spools upon which DNA is wound and packaged and also seem to be antimicrobial. Next, nuclear and granular membranes dissolve, allowing addi-tional antimicrobial proteins to attach to the DNA backbone, including crucial components of primary granules—myeloperoxidase, which generates reactive oxygen species, and neutro-phil elastase, a serine proteinase with multiple means of killing bacteria and degrading host proteins. Finally, the cell membrane is disrupted, allowing the NET to extrude into the extracellular space—the neutrophil, now dead, has secreted a web of nuclear and mito-chondrial DNA and chromatin sprinkled with antimicrobial factors that continue to capture and kill bacteria at a distance while confining the powerful NET proteins.

A variety of inflammatory stimuli that acti-vate neutrophils, including the chemokine interleukin-8, bacteria and bacterial products such as lipopolysaccharide, were believed to stimulate neutrophil receptors— predominantly CXCR1—and sequentially set off both intra-cellular killing and NETosis5 through an NADPH oxidase–dependent mechanism. CXCR1 ligation leads to activation of NADPH oxidase, which is an enzyme that catalyzes the formation of reactive oxygen species. These reactive oxygen species both mediate killing within phagolysosomes and cause membrane disruption and mixing of nuclear and granule NET components during NETosis4,5.

In cystic fibrosis, however, proteinases shed CXCR1, making CXCR2 the dominant

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neutrophil chemokine receptor. Marcos et al.2 present a convincing array of data from both human material and mouse models to show that CXCR2 signaling, independent of NADPH oxidase signaling, is responsible for NETosis in cystic fibrosis.

After showing characteristic NET structures in the airways and sputum of individuals with cystic fibrosis, they extracted neutrophils from the sputum, as well as normal neutrophils, to inhibit NET formation using antibodies and small molecules against CXCR2. They found that CXCR2-mediated NET forma-tion occurred through a Src signal transduc-tion pathway that did not involve NADPH oxidase. Moreover, NET production medi-ated by CXCR1 but not CXCR2 did not occur after inhibition of reactive oxygen species or in individuals with NADPH oxidase defi-ciency. The authors also confirmed these results in a mouse model of cystic fibrosis2. Previously, it was shown that reactive oxygen species are not sufficient to establish NETs in neutrophils of premature neonates that

have a multifactorial syndrome of neutrophil dysfunction that predisposes them to infec-tion6. Now Marcos et al.2 show that reactive oxygen species are also unnecessary.

NETosis was understood as a form of neu-trophil cell death after traditional neutrophil activation. This, however, has been chal-lenged in studies of the bloodstream7 and gastrointestinal tract8 where NETosis seems to occur in intact neutrophils rapidly upon stimulation. In a mouse model of sepsis, acti-vated platelets induced the secretion of a web from intact neutro phils within minutes that effectively captured bacteria present in the bloodstream7. Activated intact eosinophils within the gastro intestinal tract can catapult mitochondrial DNA through a reactive oxygen species–related mechanism within seconds8. Considering that 90% of total cells in humans are of bacterial origin and mostly reside within the gastrointestinal tract, it makes sense that such tight control be exerted by the gatekeeping eosinophil. Mast cells also seem to be capable of forming extra cellular traps, leading to the

term ‘ETosis’. It is not known whether these cells, which are present in cystic fibrosis lungs, contribute to ETosis.

Whereas NETs allow short-lived neutro-phils to use their antimicrobial capacity, removal of NETs presents a challenge com-pared to the process of macrophage-mediated clearance of neutrophils after apoptosis. It is known that DNases are necessary to break up NETs, yet it is unclear what the fate of NETs is. Therefore, the host may pay a price for prolonged exposure to NETs. For instance, autoantigens against NET products such as proteinase-3 and myeloperoxidase can lead to vasculitis9, endothelial cell injury and preeclampsia10.

In cystic fibrosis, NETs are ultimately inef-fective in eradicating bacteria, and the com-plications from inefficient clearance and exuberant NET debris seem to be profound. Marcos et al.2 found that NET burden corre-lated with impaired lung function in people with cystic fibrosis. They confirmed the det-rimental effect of NETs in transgenic mice with airway-specific overexpression of the amiloride-sensitive epithlelial Na+ channel11, a model that replicates airway surface liquid depletion, chronic airway neutrophilia and airflow obstruction, but not bacterial colo-nization. CFTR-deficient mice have an exag-gerated response to infection, but they do not replicate the human airway disease well. Notably, the authors also showed that applica-tion of a small-molecule inhibitor with high affinity for CXCR2 administered intranasally both inhibited NET formation and improved lung function2.

In the clinic, translation of this exciting finding to humans is certainly feasible, given the progress of the pharmaceutical industry in the development of CXCR2 inhibitors to inhibit neutrophil inflammation in a variety of diseases such as chronic obstructive pul-monary disease and ulcerative colitis. The concern associated with neutrophil and NET inhibition is that this would predis-pose individuals to severe infection. In the case of cystic fibrosis, local administration would limit systemic effects. Nevertheless, the lung is exactly the place where the daily battle between the host and microbes resides in people with this disease.

Further work on the effect of CXCR2 inhibi-tion on the microbial flora in cystic fibrosis is needed. It is encouraging, however, that inhi-bition of CXCR2 may prevent NET formation and loss of lung function in people suffering from cystic fibrosis. Of course, it would also still be ideal to correct the genetic defect to eliminate all downstream effects of CFTR mutations in cystic fibrosis.

Figure 1 NEts contribute to airway obstruction in cystic fibrosis. the airway of individuals with cystic fibrosis is characterized by the presence of thick, sticky sputum, chronic infection with P. aeruginosa and chronic neutrophilic inflammation. the CXCR1 receptor is cleaved in cystic fibrosis by proteinases such as neutrophil elastase (NE). Marcos et al.2 now show that interleukin-8 (CXCL8), and perhaps other CXC chemokines, can bind the receptor CXCR2 on neutrophils, leading to the formation of NEts in a cystic fibrosis mouse model and in individuals with cystic fibrosis. this pathway is mediated by Src tyrosine kinase but is independent of NADPH oxidase. NEts are composed of a chromatin backbone with antimicrobial agents such as histones, NE and myeloperoxidase (MPO). the inefficient clearance of NEts results in an even more viscous mucus that worsens the airflow in cystic fibrosis and can aggravate lung function. inhibition of CXCR2 with antibodies or small-molecule inhibitors prevents NEt formation, decreasing neutrophilic debris in the airway and allowing apoptotic neutrophils to be taken up by macrophages.

Large conducting airway of cystic fibrosis

Inspissated sputum

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Thicker mucus causes airwayobstruction and worsens lung function

Blocking NETs production improves lungfunction with cleaner airways and less mucusK

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Despite these promising developments, some questions remain unanswered. As the authors focused on ALDH-2i, they did not determine whether disulfiram itself increases THP levels, nor did they examine the behavioral effects of disulfiram in parallel with ALDH-2i4, making it difficult to draw direct conclusions about the clinical efficacy of disulfiram. In fact, ALDH-2i reduces tissue dopamine amounts, whereas disulfiram actually increases dopamine abundance, at least in the cortex5,6.

What’s more, it is unclear whether the effects of ALDH-2i are specific to cocaine or extend to general operant processes, such as lever press-ing for a natural reward such as food. The authors showed that ALDH-2i had no effect on the number of presses on the inactive lever, which was not paired with cocaine infusions, but this is not an ideal control, as the number of inactive lever presses is very low, potentially creating a ‘floor effect’ that can mask reduc-tions in lever pressing. It would therefore be interesting to test whether ALDH-2i affects operant and reinstatement responding for a nondrug reinforcer, such as sucrose. Despite these limitations, this exciting study provides compelling evidence for ALDH-2 inhibition as a mediator of disulfiram-induced reductions in cocaine use and as a new therapeutic strategy.

Although this study provides an appeal-ing explanation for how disulfiram curbs cocaine use, additional mechanisms may be in play. Because cocaine use increases the amount of norepinephrine in the brain as well as the amount of dopamine, it has been suggested that the ability of disulfiram to inhibit dopamine β-hydroxylase (DBH), an enzyme required for norepinephrine produc-tion in brainstem noradrenergic neurons, is what underlies its clinical efficacy in cocaine dependence7,8.

Despite decades of research, we still lack therapies for the treatment of cocaine addic-tion. There is, however, a treatment for alcoholism—the drug disulfiram (Antabuse, Odyssey Pharmaceuticals) inhibits the enzyme aldehyde dehydrogenase (ALDH), which is responsible for the breakdown of the alcohol metabolite acetaldehyde. Blocking ALDH results in the accumulation of acetaldehyde, which is toxic, and leads to a series of aversive symptoms that deter alcohol consumption1.

Given the comorbidity of cocaine and alcohol abuse, a team of psychiatrists led by Kathleen Carroll in the early 1990s specu-lated that discouraging alcohol use with disul firam may also reduce cocaine intake. The results of several clinical trials confirmed the efficacy of disulfiram in the treatment of cocaine dependence2.

But even before the first congratulations were heard, a closer look at the data revealed the efficacy of disulfiram did not depend on alcohol intake, suggesting the existence of another mechanism different from the one dependent on the accumulation of acetalde-hyde. This is crucial, as disulfiram is not a ‘magic bullet’; its efficacy is moderate and it has several harsh side effects, such as hepato-toxicity, that limit its potential use. Unveiling its specific mechanisms of action could pave the way for more potent and selective com-pounds as therapeutics for addiction.

The addictive properties of cocaine are mediated by its ability to block the trans-porters that limit extracellular amounts of the neuro transmitters dopamine, norepi-nephrine and serotonin in the brain. It was

previously shown that a selective inhibitor of ALDH-2, ALDH-2i, reduces alcohol intake in rats by preventing alcohol-induced dopamine release, even in the absence of acetaldehyde3. This raised a question of whether the reduc-tion of cocaine use caused by disulfiram might be mediated by a similar mechanism. In this issue of Nature Medicine, Yao et al.4 unveil how inhibition of ALDH-2 can suppress self-administration of cocaine and reinstate-ment of cocaine seeking in rats. The authors show that blocking of ALDH-2 results in increased amounts of tetra hydropapaveroline (THP) in neurons in the midbrain, which, in turn, inhibits production and reduces levels of dopamine.

Using operant cocaine self-administration, in which rats are trained to press a lever to receive cocaine, the authors found that ALDH-2i sup-presses drug intake4. ALDH-2i also attenu-ated two models of relapse—the ability of an experimenter-delivered cocaine injection or tone and light cues paired with cocaine deliv-ery to reinstate lever pressing—after the drug-seeking behavior disappeared. Yao et al.4 then began the search for a mechanism to explain the behavioral effects of ALDH-2i.

ALDH-2 catalyzes the conversion of the dopamine metabolite 3,4-dihydroxyphenyl-acetaldehyde (DOPAL) to 3,4-dihydroxyphenyl-acetic acid (DOPAC). Blocking this reaction results in the accumulation of DOPAL, which can combine with dopamine to form THP. THP is a potent inhibitor of tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis, and the authors show that either ALDH-2i or THP itself can block cocaine-induced increases in dopamine production and reinstatement of cocaine seeking4. They therefore concluded that inhibiting ALDH-2 reduces cocaine intake and reinstatement by attenuating dopamine production and transmission via THP4.

David Weinshenker is in the Department of Human

Genetics, Emory University School of Medicine,

Atlanta, Georgia, USA.

e-mail: dweinsh@emory.edu

Cocaine sobers upDavid Weinshenker

A drug that discourages alcohol ingestion has shown promise as a treatment for cocaine addiction. New findings in rats suggest a potential mechanism—the drug decreases amounts of dopamine in the brain (pages 1024–1028). Blocking enzymes that regulate dopamine abundance may be a new way to treat cocaine addiction and prevent relapse in humans.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

1. Walters, S. & Mehta, A. in Cystic Fibrosis 3rd edn. (eds. Hodson, M., Geddes, D.M. & Bush, A.) 21–45 (Hodder Arnold, London, 2007).

2. Marcos, V. et al. Nat. Med. 16, 1018–1023 (2010).

3. Downey, D.G., Bell, S.C. & Elborn, J.S. Thorax 64, 81–88 (2009).

4. Brinkmann, V. et al. science 303, 1532–1535 (2004).5. Brinkmann, V. & Zychlinsky, A. Nat. Rev. Microbiol. 5,

577–582 (2007).6. Yost, C.C. et al. Blood 113, 6419–6427 (2009).7. Clark, S.R. et al. Nat. Med. 13, 463–469 (2007).

8. Yousefi, S. et al. Nat. Med. 14, 949–953 (2008).9. Kessenbrock, K. et al. Nat. Med. 15, 623–625 (2009).10. Gupta, A.K., Hasler, P., Holzgreve, W., Gebhardt, S. &

Hahn, S. Hum. immunol. 66, 1146–1154 (2005).11. Mall, M., Grubb, B.R., Harkema, J.R., O’Neal, W.K. &

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