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Role of TNF- in sepsis

During sepsis, activation of proinflam-matory pathways leads to dysfunctionof mitochondria and cells, which con-tributes to multiorgan failure and pooroutcomes. TNF- simultaneously acti-

vates the JNK and NF-kB pathways andincreases expression of the mitochon-drial antioxidant protein SOD2, leading

to an increase in H2O2 and the inactiva-tion of JNK phosphatases. TNF recep-tors are differentially expressed in vari-ous cells and tissues and play a key rolein modulating the cellular inflamma-tory responses (1). The severity of injuryto the pulmonary endothelium duringsepsis is determined by a complex inter-play among proinflammatory cytokines

(including TNF-), adhesion moleculesexpressed on the endothelial cells, andleukocytes recruited to the site of injury.Overwhelming sepsis or perturbation ofthe adaptive responses may lead to exacer-bation of inflammation (2). An importantmechanism to limit the TNF-mediatedproinflammatory response involves shed-ding of the TNF- receptor 1 (TNFR1) ecto-

domain, which is controlled by TNF-converting enzyme (TACE; also knownas ADAM17), a disintegrin metallo-proteinase (3, 4). Here, Rowlands et al.provide evidence that in the lung micro-

vascular endothelium, administrationof soluble TNF- (sTNF-) increasedcytosolic levels of Ca2+ via inositol-1,4,5-triphosphatemediated (IP3-medi-ated) release from the ER (5). The increasein cytosolic Ca2+ was followed by a rapidincrease in mitochondrial Ca2+, leading toa rise in mitochondrial ROS generation

from complex III of the electron trans-port chain. These ROS were required for

TACE-mediated shedding of the TNFR1ectodomain, which in turn modulated theendothelial inflammatory response.

Mitochondrial function and Ca2+

signaling

Ca2+ uptake by mitochondria is highlyregulated, and the levels of mitochondrialCa2+ play a central role in the metabolic(ATP production) and signaling (ROSproduction, release of proapoptotic mol-

ecules) functions of the organelle (Figure1A). In patients with acute sepsis, mito-chondrial function is impaired (Figure1B), and in patients with septic shock,the severity of impairment in mitochon-drial function is associated with adverseclinical outcomes (6, 7). Normalizationof mitochondrial biogenesis is necessaryto restore oxidative metabolism duringsepsis (Figure 1C) so that adequate O2delivery and O2 tissue consumption canresume, allowing cellular metabolic needsto be met (8, 9).

Rowlands et al. suggest that the TNF-mediated increase of mitochondrial Ca2+is dependent on ER Ca2+ release leadingto IP3-mediated depletion of intracellularstores (5). It is known that store deple-tion triggers the activation of an inwardrectifying Ca2+ current mediated eitherby the action of specialized Ca2+ releaseactivated (CRAC) channels or by theopening of voltage, receptor, or secondmessenger operated channels. TNF-induced increases in cytosolic Ca2+ leadto increased mitochondrial Ca2+ uptake

through mitochondrial voltage-depen-dent anion channels, the mitochondrial

Mitochondrial Ca2+ and ROS take center stage

to orchestrate TNF-mediated

inflammatory responsesLaura A. Dada and Jacob I. Sznajder

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Proinflammatorystimuliinduceinflammationthatmayprogresstosep-sisorchronicinflammatorydisease.ThecytokineTNF-isanimportantendotoxin-inducedinflammatoryglycoproteinproducedpredominantlybymacrophagesandlymphocytes.TNF-playsamajorroleininitiat-ingsignalingpathwaysandpathophysiologicalresponsesafterengagingTNFreceptors.InthisissueofJCI,Rowlandsetal.demonstratethatinlungmicrovessels,solubleTNF-(sTNF-)promotesthesheddingoftheTNF-receptor1ectodomainviaincreasedmitochondrialCa2+thatleadstoreleaseofmitochondrialROS.SheddingmediatedbyTNF- convert-ingenzyme(TACE)resultsinanunattachedTNFreceptor,whichpartici-patesinthescavengingofsTNF-,thuslimitingthepropagationofthe

inflammatoryresponse.ThesefindingssuggestthatmitochondrialCa2+,ROS,andTACEmightbetherapeuticallytargetedfortreatingpulmonaryendothelialinflammation.

Conflictofinterest:J.I. Sznajder is the editor of theAmerican Journal of Respiratory and Critical Care Medicineand receives a stipend from the American Thoracic Soci-ety for this function.

Citationforthisarticle: J Clin Invest. doi:10.1172/JCI57748.

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Ca2+ uniporter (MCU) and rapid modeof uptake channels. This process alsoinvolves the high-conductance permea-bility transition pore (PTP), which spansthe outer and inner mitochondrial mem-brane and can allow rapid ion transfer,leading to mitochondrial swelling anddysfunction. The molecular Ca2+ signal-ing machinery is complex, and studiesinto the role played by the many channels

involved has been limited by incompleteidentification of the proteins compris-ing them, limiting the development ofgenetic or specific pharmacologic tools.Instead, investigators have relied on stud-ies using relatively nonspecific inhibitors,for example, ruthenium red to inhibitMCU and cyclosporine A to inhibit thePTP. Interpretation of these studies iscomplicated by the off-target effects ofthese agents. Recently, substantial prog-ress has been made in identifying the crit-ical protein constituents of these chan-

nels. For example, Perrochi et al. recentlyidentified the mitochondrial calcium

uptake 1 (MICU1) channel, providing anovel molecular target to prevent mito-chondrial Ca2+ overload (10).

Mitochondrial Ca2+ and ROS

signaling in response to TNF

During oxidative phosphorylation, elec-trons from reduced substrates are trans-ferred to O2 through a chain of electrontransporters, and the energy provided

by these electron transfer reactions isused to transfer protons across the mito-chondrial inner membrane. The energystored in the resulting chemiosmoticgradient is used to generate ATP. Duringelectron transfer, some electrons reactwith molecular O2 to produce a super-oxide anion that is converted to H2O2by mitochondrial or cytosolic SODs.Complexes I and II of the electron trans-port chain release ROS exclusively in themitochondrial matrix, whereas complexIII generates ROS on both sides of the

mitochondrial inner membrane (11).Influx of Ca2+ accelerates the generation

of reduced substrates from the TCA cycleand the rate of several reactions in theelectron transport chain, including O2consumption and ROS generation. Ca2+also increases the activity of NO syn-thase, elevating NO levels, which in turninhibit cytochrome oxidase (complex IV)and might also increase the productionof ROS. Although classically consideredtoxic byproducts of cellular respiration,

more recent evidence suggests that ROSreleased at moderate levels may activatecytosolic signaling pathways and tran-scriptional programs, allowing the cell toadapt to environmental stress (12).

The increase in mitochondrial Ca2+leads to ATP and ROS production,which can trigger additional increases inCa2+ as a result of further extracellularCa2+ influx (1315). This represents atipping point in cell survival: low lev-els of ROS generated at complex III canactivate adaptive signaling pathways, but

higher levels of ROS result in the releaseof cytochrome c and cell death (12, 16).

Figure 1Role of mitochondrial Ca2+ and TACE in modulating inflammation of the pulmonary endothelium. (A) During regulated inflammatory response,

sTNF- increases mitochondrial Ca2+ and ROS, leading to TACE-mediated TNF receptor ectodomain shedding in endothelial cells. (B) During

overwhelming sepsis, increased Ca2+ influx into the mitochondria leads to its dysfunction and to cell death. (C) Restoring the mitochondrial func-

tion by decreasing Ca2+ influx into the mitochondria may be a potential therapeutic target during sepsis.

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commentaries

In a series of elegant experiments, Row-lands et al. transfected several custom-designed fluorophore probes into mouselung microvascular endothelium; con-focal microscopy then showed that the

intracellular Ca2+

increase occurs in twosteps (5). The initial increase correspondswith sTNF- activation of the lungmicrovascular endothelium and is asso-ciated with expression of the leukocyteadhesion receptor E-selectin and TACE-mediated TNF receptor shedding. Thisinitial spike is followed by prolongedelevation of Ca2+ that persists even whenTNF- infusion is stopped and is medi-ated by the purinergic receptor P2Y2 (5).

TNF receptor shedding as an

antiinflammatory adaptation

response

Ectodomain TNF receptor shedding isan important mechanism of adaptationand/or protection against inflamma-tion. The unanchored receptor bindsthe soluble TNF, decreasing its ability totransduce intracellular signals, and thuscurtails the cytokines proinflammatoryeffects. TNF receptor inhibition is anaccepted therapeutic modality in manyinflammatory diseases, and the TNF-inhibitors etanercept (a recombinantsoluble fusion protein consisting of TNF

receptor coupled to the Fc portion ofIgG), adalimumab (a human antiTNF-antibody), and infliximab (a chimericIgG1 antiTNF- antibody) are widelyused therapeutics (17). An impaired abil-ity to shed or endocytose TNF receptorscan lead to inflammatory diseases. Forexample, a mutation in the gene encodingTNFR1 that impairs receptor sheddingcauses TNF receptorassociated periodicsyndrome (TRAPS), an autosomal-domi-nant autoinflammatory disease (18).Consistent with the findings of Rowlands

et al. (5), a protective role of TNFR1 shed-ding by TACE was also reported in TNF-sensitization of hepatocytes to Fas-medi-ated death (19).

Relevance to sepsis

Sepsis remains a common clinical prob-lem in the United States; therapeuticoptions to address the resulting multior-gan failure are limited, and the mortalityin patients with sepsis remains unaccept-ably high. Sepsis occurs in patients withoverwhelming infections and leads to the

inability of cells to use the O2 deliveredto them. In patients with septic shock,

cardiac output is normal or even supra-normal, and the delivery of O2 to tissuesafter acute resuscitation is normal orincreased. However, the response to infec-tion in patients with sepsis can result in

impaired mitochondrial function, whichmay contribute to cell death and multi-organ dysfunction (Figure 1B and refs. 8,20). We reason that in severe sepsis, thereis a massive influx of Ca2+, part of which istaken up by mitochondria via the above-described Ca2+ channels. This rapid influxof Ca2+ leads to swelling and mitochon-drial dysfunction, which, in conjunctionwith proinflammatory TNF signaling,contributes to cell death.

Rowlands et al. provide evidence thatcontrolled TNF infusion leads to sus-

tained mitochondrial Ca

2+

elevation andmitochondrial ROS generation in thepulmonary endothelium (5). At the levelsthey observed, these ROS-activated sig-naling pathways resulted in TNF recep-tor shedding via TACE, thereby limitingthe inflammatory response (Figure 1A).

A limitation of this study and of mostresearch in this area is that researchersuse a model of moderate sepsis to eluci-date mechanisms underlying the responseto TNF-. It is tempting to speculate thathigher levels of sTNF- in more severe sep-sis would lead to influx of Ca2+ and that

these same pathways would cause mito-chondrial dysfunction and perhaps death.

AntiTNF- therapy has been ineffec-tive in the treatment of sepsis and criti-cal illness, but has been used with successin rheumatoid arthritis, Crohn disease,ankylosing spondylitis, and other chronicinflammatory diseases. This therapy hasbeen associated with potentially severeside effects, including tuberculosis andserious bacterial and opportunistic infec-tions. The pathways Rowlands et al. haveidentified (5) and the recent discovery of

MICU1 protein (10) are encouraging andhighlight mechanisms that might be tar-geted to ameliorate the inflammation andorgan dysfunction observed in patientswith sepsis (Figure 1C).

Acknowledgments

The authors are supported by NIH grantsR37-HL48129, HL-85534, and PO1-HL71643.

Address correspondence to: JacobI. Sznajder, Pulmonary and Critical

Care Medicine, Northwestern Univer-sity, Feinberg School of Medicine, 240

E. Huron, McGaw M-300, Chicago, Illi-nois 60611, USA. Phone: 312.908.7737;Fax: 312.908.4650; E-mail: j-sznajder@northwestern.edu.

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