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Am J Physiol Lung Cell Mol Physiol 306: L962–L974, 2014.
First published April 18, 2014; doi:10.1152/ajplung.00073.2014.
Perspectives
Mitochondria in lung biology and pathology: more than just a powerhouse
Paul T. Schumacker,1 Mark N. Gillespie,2 Kiichi Nakahira,3 Augustine M. K. Choi,3 Elliott D. Crouser,4
Claude A. Piantadosi,5 and Jahar Bhattacharya6
1
Northwestern University Feinberg School of Medicine, Department of Pediatrics, Chicago, Illinois; 2University of South
Alabama College of Medicine, Department of Pharmacology, Mobile, Alabama; 3Weill Cornell Medical College, Department
of Medicine, New York, New York; 4The Ohio State University College of Medicine, Department of Internal Medicine,
Columbus, Ohio; 5Duke University School of Medicine, Department of Medicine, Durham, North Carolina, and 6Columbia
University Medical Center, Department of Physiology and Cellular Biophysics, New York, New York
Submitted 18 March 2014; accepted in final form 9 April 2014
hypoxia; lung injury; mitochondrial biogenesis; mitochondrial transfer; mtDNA
“THE POWERHOUSE OF THE CELL.” All of the contributors to the
2013 ATS symposium forming the basis of this Perspectives
article, and perhaps many of the attendees, too, recalled this
phrase as their first introduction to mitochondria and often their
first memorable phrase in biology. Though still true, it now
seems almost quaint given the explosion of information about
mitochondrial functions unrelated to energy metabolism that
has occurred over the past two decades or so. Our symposium
was motivated partly by this “explosion” and partly by the
equally rapid accumulation of evidence that mitochondria
likely play crucial roles in lung disease with significant potential to serve as targets in pulmonary and critical care medicine.
This Perspectives article does not seek to comprehensively
review all developments in the mitochondrial field that bear on
lung disease. Rather, it highlights evolving concepts of how
mitochondria contribute to normal lung health and how mitochondria could be targeted for specific intervention in lung
disease. Having registered this, we present concise summaries
pertaining to mitochondria, reactive oxygen species (ROS),
and oxygen sensing in the lung, the role of the mitochondrial
genome as a sentinel molecule governing lung cell survival in
response to oxidant stress, the nature of mitochondrial DNA
Address for reprint requests and other correspondence: M. N. Gillespie,
Dept. of Pharmacology, College of Medicine, Univ. of South Alabama,
Mobile, AL 36688 (e-mail: [email protected]).
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(mtDNA) damage-associated molecular patterns (DAMPs)
originating from the lung, and their potential use and importance as biomarkers and proinflammatory alarm signals. Finally, we explore the intriguing notion that therapeutic strategies aimed at reconstituting normal mitochondrial population
and function may comprise therapeutic approaches in acute
lung injury (ALI).
Mitochondrial Oxygen Sensing
Mitochondria consume O2 at cytochrome c oxidase, the
terminal complex in the electron transport chain (ETC). Electron flux along the ETC is linked to proton translocation across
the inner mitochondrial membrane, resulting in the generation
of an electrochemical gradient that is used by the ATP synthase
to generate ATP during oxidative phosphorylation. As mitochondria convert O2 to water the intracellular O2 tension
decreases, thereby generating a gradient for O2 diffusion into
the cell from the extracellular space. Mitochondria are the
principal site of oxygen consumption in the cell, so their
oxygen tension is lower than that of any other organelle. This
characteristic has led investigators to ask whether mitochondria
function as oxygen sensors in the cell (64). A conceptually
simple idea is that decreases in cellular O2 levels (tissue
hypoxia) would limit mitochondrial oxygen consumption,
leading to redox changes as the ETC complexes became
saturated with electrons owing to the loss of cytochrome c
oxidase activity (56). These redox changes would then be
reflected in the redox status of systems that feed electrons into
the ETC, such as the Krebs cycle. However, a conceptual
problem with this idea arose from experimental studies showing that mitochondria do not become limited by oxygen availability until the cell is nearly anoxic (12, 95). Specifically, how
could the mitochondria respond to physiological levels of
hypoxia if their ability to signal via redox alterations was not
activated until all of the oxygen was gone? For many years this
paradox represented a conceptual barrier to the idea that
mitochondria might be involved in oxygen sensing. Some
investigators suggested that specialized oxygen sensors such as
the glomus cells of the carotid body might circumvent this
problem by expressing special isoforms of cytochrome c oxidase with low apparent affinity for O2 (59). In those cells,
electron transport and oxygen consumption would become O2
supply limited at milder levels of hypoxia, allowing the mitochondria to detect subtle decreases in cellular oxygenation
(58). However, no experimental evidence for the existence of
such a low-affinity oxidase has been reported. Moreover,
mammalian cells with high-affinity forms of cytochrome c
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Schumacker PT, Gillespie MN, Nakahira K, Choi AM, Crouser
ED, Piantadosi CA, Bhattacharya J. Mitochondria in lung biology
and pathology: more than just a powerhouse. Am J Physiol Lung Cell
Mol Physiol 306: L962–L974, 2014. First published April 18, 2014;
doi:10.1152/ajplung.00073.2014.—An explosion of new information
about mitochondria reveals that their importance extends well beyond
their time-honored function as the “powerhouse of the cell.” In this
Perspectives article, we summarize new evidence showing that mitochondria are at the center of a reactive oxygen species (ROS)dependent pathway governing the response to hypoxia and to mitochondrial quality control. The potential role of the mitochondrial
genome as a sentinel molecule governing cytotoxic responses of lung
cells to ROS stress also is highlighted. Additional attention is devoted
to the fate of damaged mitochondrial DNA relative to its involvement
as a damage-associated molecular pattern driving adverse lung and
systemic cell responses in severe illness or trauma. Finally, emerging
strategies for replenishing normal populations of mitochondria after
damage, either through promotion of mitochondrial biogenesis or via
mitochondrial transfer, are discussed.
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Fig. 1. Conceptual model of oxygen sensing by mitochondria through the
regulated generation of reactive oxygen species (ROS) that are released to the
cytosol through the intermembrane space (IMS). IMS-PRDX5, peroxiredoxin-5 targeted to the IMS; PHD, hypoxia-inducible factor (HIF) prolyl
hydroxylase; III, mitochondrial complex III.
intracellular ROS levels and pharmacological inhibitors to
block the ETC (34, 90). Importantly, different laboratory
groups using the same inhibitors at various concentrations
reported conflicting responses. To address this, Waypa et al.
(91) utilized a novel genetically encoded fluorescent protein
sensor to detect intracellular ROS levels. The initial sensor,
HSP-FRET, exhibited ratiometric behavior that obviated an
important limitation of the fluorescent chemical probes. Specifically, ratiometric sensors provide a signal that is independent of the light intensity used to excite the probe. Importantly,
studies using HSP-FRET expressed in pulmonary artery
smooth muscle cells confirmed the previous observation that
ROS levels increase in the cytosol during hypoxia (91). Moreover, the increase in ROS during hypoxia was shown to be
required for the increase in cytosolic Ca2⫹ activation in pulmonary artery smooth muscle cells. Further advances in this
hypoxia-induced ROS signaling model came with the introduction of roGFP, a redox-sensitive mutant of green fluorescent protein (24, 35). This ratiometric sensor provides a specific measure of protein thiol oxidation/reduction status in the
cell. Two important features contributed to the importance of
this advance. First, its oxidation is reversible in the cell,
allowing it to be calibrated, analogous to the behavior of
ratiometric Ca2⫹ sensors. Second, its expression can be targeted to specific intracellular compartments, allowing assessment of redox at the subcellular level. Using roGFP expressed
in cytosol, mitochondrial intermembrane space (IMS), and the
mitochondrial matrix, Waypa et al. (92) found that significant
differences in basal oxidant status exist across these compartments in pulmonary artery smooth muscle cells (PASMC),
with the cytosol existing in a more reduced state, the mitochondrial matrix in a highly oxidized state, and the IMS at an
intermediate level. During acute hypoxia, oxidation status in
the IMS and the cytosol increased, whereas oxidation in the
mitochondrial matrix decreased. These findings were consistent with a model in which the release of superoxide from
complex III to the IMS increases during hypoxia, allowing the
ROS to potentially exit to the cytosol and initiate redox
signaling involved in triggering the release of intracellular
calcium stores (94). By contrast, hypoxia caused an attenuation
of oxidant stress in mitochondrial matrix. The opposing redox
responses to hypoxia in these two compartments could reflect
a distinct operation of two independent mechanisms controlling ROS generation during hypoxia in the two domains.
Alternatively, the possibility that ROS alters the direction of
ROS secretion from the membrane, toward the matrix during
normoxia and toward the IMS during hypoxia, has not been
ruled out.
In either case, studies were needed to more critically test the
hypothesis that the mitochondrial electron transport chain is the
source of ROS signaling during hypoxia. Accordingly, Waypa
et al. (93) developed a genetic model by generating a mouse to
permit conditional deletion of the Rieske iron-sulfur protein
(RISP). This nuclear encoded protein is critical for the function
of complex III, where it accepts an electron from ubiquinol, the
carrier that moves electrons into complex III from complexes
I and II (34). ROS generation from complex III can occur after
RISP accepts an electron from ubiquinol, generating the intermediate free radical, ubisemiquinone. The remaining electron
on ubisemiquinone is normally transferred to the b-cytochromes in the complex but can potentially be transferred
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oxidase are still capable of detecting and responding to hypoxia.
A second hypothesized model of oxygen sensing was promoted by Michelakis, Archer, and Weir, who sought to explain
how pulmonary artery smooth muscle cells detect hypoxia and
trigger acute hypoxic pulmonary vasoconstriction (4). Their
model proposed the existence of an intracellular oxidase that
would generate more ROS at high O2 levels and less under
hypoxic conditions. According to that model, ROS would then
activate voltage-dependent potassium (Kv) channels in the
plasma membrane. Conversely, the lesser oxidation of these
channels during hypoxia would promote channel closure, causing membrane depolarization that would lead to the entry of
extracellular Ca2⫹ through L-type calcium channels. However,
when they tested the effect of gp91-(phox) deletion on this
response, using mice with genetic deletion of NOX2, they
found that the cellular response to hypoxia was still present (3).
They subsequently considered that mitochondria were the
source of these ROS signals, based on studies using various
mitochondrial inhibitors (57). However, a conceptual problem
with that model has been the consistent failure of diverse
chemical antioxidants or the expression of enzymatic antioxidant systems to activate hypoxic responses in a normoxic cell.
A third model of mitochondrial oxygen sensing (Fig. 1)
arose from initial studies showing that intracellular ROS levels
actually increase during hypoxia (13, 26). Parallel studies using
chemical mitochondrial inhibitors implicated complex III as
the likely source of ROS generation. This paradoxical response
was initially criticized because it seemed counterintuitive and
was based on the use of nonspecific fluorescent probes to detect
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cells also showed a significant attenuation of the acute hypoxiainduced activation of cytosolic Ca2⫹. These findings provide an independent demonstration that ROS release from
the mitochondrial inner membrane to the IMS and subsequently to the cytosol is critical for triggering acute responses to hypoxia in PASMC.
Previous studies by this group had shown that the stabilization of hypoxia-inducible factor-1␣ (HIF-1␣) is triggered by
hypoxia-induced release of ROS from mitochondria. In
PASMC, hypoxia also triggers stabilization of HIF-1␣ by
inhibiting prolyl hydroxylase, which targets the protein for
degradation by hydroxylating conserved proline residues during normoxia (38, 41). In PASMC expressing graded levels of
PRDX5 in the IMS, hypoxia-induced HIF-1␣ stabilization was
inhibited in a dose-dependent manner (79).
Collectively, these findings indicate that mitochondria function as more than simple factories supplying ATP to the cell.
Through the regulated release of ROS to the cytosol, these
organelles have the capacity to orchestrate a diverse group of
adaptive responses to hypoxia. In that regard, the moniker of
“cellular oxygen sensors” clearly applies to these organelles.
Sentinel Functions of mtDNA in Pulmonary Vascular Cells
The nuclear and mitochondrial genomes differ in certain
fundamental respects. For example, compared with the nuclear
genome, the mitochondrial genome is tiny. Even if all the
mtDNA molecules in cells with high mitochondrial densities
are totaled, it is still a small fraction of the size of the nuclear
genome. Following from this, mtDNA encodes for only a
handful of proteins compared with the many thousands encoded by nuclear DNA. Second, the structure and regulation of
the mitochondrial genome is comparatively simple. Unlike
nuclear DNA, in which the state of compaction, its interactions
with multiple transcription factors and coactivators, and epigenetic determinants are all exquisitely regulated processes
culminating in precise control of gene expression, the mitochondrial genome is expressed as a single unit and its transcription is controlled by a handful of mtDNA binding proteins,
particularly mitochondrial transcription factor A (TFAM) (10,
44). Instead of compaction within a nucleosome context, a
single molecule of mtDNA is associated with ⬃30 core proteins in a structure known as the nucleoid. Most if not all of the
enzymes needed for mtDNA replication and mtRNA expression appear to be components of the nucleoid (9).
Another difference between mitochondrial and nuclear DNA
particularly relevant to the present discussion is that mtDNA is
⬃50-fold more sensitive to oxidative damage (7, 16, 32, 97).
Reasons for this heightened sensitivity are not entirely known,
but have been attributed to a comparatively simple process of
mtDNA repair (mitochondria seem to contain only base excision repair whereas the nucleus has multiple, overlapping
pathways) (8), and the prospect that the open structure of
mtDNA is more accessible to DNA-damaging ROS. In light of
the forgoing comments that mitochondrial ROS generation
plays a central role in hypoxic signal transduction, the question
can be raised as to whether mtDNA is damaged by oxidation in
hypoxic environments. Although DNA integrity across the
entire mitochondrial genome in hypoxic cells has not been
examined, a large expanse of the mtDNA coding region,
including the “common deletion sequence” known to be sen-
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instead to O2, generating superoxide. If RISP is deleted,
ubisemiquinone, and thus superoxide, cannot be generated at
complex III (33, 54). Accordingly, that group studied PASMC
isolated from the conditional RISP mice and used Cre recombinase expression to delete the RISP gene. After loss of RISP
was confirmed, subsequent roGFP studies demonstrated that
the hypoxia-induced changes in redox status in the cytosol,
IMS, and the mitochondrial matrix were abolished. These
studies therefore demonstrated that a functional complex III
was required for the changes in redox observed in different
subcellular compartments during hypoxia (93). In parallel
studies, it was found that the transient increase in cytosolic
Ca2⫹ in PASMC during acute hypoxia was abrogated in cells
where RISP had been deleted (93).
A technical limitation of studies using cultured PASMC is
the possibility that the cells will undergo a phenotypic shift in
culture. Moreover, studies in cultured cells cannot provide an
assessment of the role of those responses in the intact lung.
Accordingly, that group conducted in vivo studies in which
RISP was deleted from smooth muscle cells by breeding the
conditional RISP mice with a transgenic line expressing a
smooth muscle-specific, tamoxifen-inducible Cre (96). The
resulting mice were administered tamoxifen to activate nuclear
localization of the Cre in smooth muscle cells, and subsequent
deletion of RISP from smooth muscle was confirmed. The
acute pulmonary vasoconstriction response was then compared
in the knockout and control mice, by measuring the right
ventricular systolic pressure (RVSP) response to acute alveolar
hypoxia. Control mice exhibited a significant increase in RVSP
during 5% O2 ventilation, which was significantly attenuated in
the RISP-deficient mice (93). These findings indicate that a
functional complex III is required for the acute vasopressor
response to hypoxia in the lung.
Does the same mitochondrial ROS signal trigger other functional responses to hypoxia? Mungai et al. (60) tested the
hypothesis that hypoxia activates the cellular energy sensor,
AMP-dependent kinase (AMPK), through an ROS-dependent
mechanism. In PASMC, they found that ROS generation
during moderate hypoxia led to AMPK activation, even in
the absence of any increase in AMP or ADP concentrations.
Those findings suggest that mitochondrial ROS signals activate
AMPK as an early response to hypoxia, allowing it to activate
protective mechanisms before the cell progresses into a bioenergetic crisis where AMP and ADP concentrations are increased.
Do specific antioxidants block these responses? To test this,
Sabharwal et al. (79) reasoned that the expression of an
enzymatic scavenger of H2O2 exclusively in the mitochondrial
IMS should abolish functional responses to hypoxia by preventing ROS released from complex III to reach the cytosol.
Peroxiredoxin-5 (PRDX5) is an atypical 2-cysteine-containing
peroxidase that is normally expressed in multiple subcellular
compartments including the mitochondrial matrix (45). This
enzyme functions as a monomer, whereas other H2O2 scavengers only function as multimers. Sabharwal et al. targeted its
expression to the IMS using the NH2-terminal sequence that
directs the SMAC/Diablo protein to that compartment (66) and
confirmed correct localization using immunogold electron microscopy (79). When PRDX5 was expressed in the IMS of
PASMC, it attenuated the increase in ROS in the IMS and the
cytosol during hypoxia. Importantly, the PRDX5-expressing
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oxidatively damaged mtDNA or mtDNA damage-related proteins. In this regard, overexpression of a mitochondrially targeted DNA repair-defective Ogg1 mutant protected against
asbestos-induced apoptosis in A549 cells (67). Because the
repair-defective enzyme, like the wild-type enzyme, bound
aconitase, the possibility was advanced that Ogg1 influences
ROS-mediated signaling events downstream of mtDNA damage (80). In a related context, it has recently been shown that
Ogg1 in complex with free 8-oxoguanine serves as a guanine
exchange factor to activate Ras-mediated signaling (29), but
whether this occurs in intact cells with ROS-induced mtDNA
damage has not been established.
As a means of exploring involvement of oxidative mtDNA
damage in intact lung and animal models of pulmonary disease, Wilson and colleagues (48) developed novel fusionprotein constructs targeting DNA repair glycosylases, either
Ogg1 or the bacterial enzyme Endo III, to mitochondria. Early
work showed that these fusion protein constructs prevented
both hydrogen peroxide-induced mtDNA damage and increases in the vascular filtration coefficient in isolated perfused
rat lungs (16). The Ogg1 construct also attenuated ventilatorinduced lung injury and mortality in mice (36) and hyperoxiainduced dysmorphogenesis in fetal rat lung explants (28).
Preliminary studies also suggest that mitochondrially targeted
Ogg1 inhibits mortality and ALI evoked by intratracheal Pseudomonas aeruginosa in rats (15).
These observations on the effectiveness of increasing
mtDNA repair in isolated lungs and animal models are a step
toward determining whether mtDNA integrity is a legitimate
pharmacological target in lung disease. However, there are
many unresolved issues. As noted above, some pertain to the
specific mechanism whereby increased mtDNA repair protects
against cytotoxicity. As an extension of this, whereas protection of mtDNA integrity in cultured cells inhibits ROS-induced
cytotoxicity, the involvement of cytotoxicity per se in intact
animal models of ALI is complex. Taking this analysis one
step further, it is easy to envision how oxidative mtDNA
damage could, by different mechanisms, mediate different
types of cellular responses in lung disease. For example,
ROS-induced cytotoxicity, driven by persistent mtDNA damage, defective mtDNA transcription, and attendant bioenergetic
insufficiency, could be lethal to some populations of lung cells,
whereas oxidant-induced alterations in mtDNA-protein stability might acutely influence behavior of other lung cell populations secondary to alterations in cytoskeletal integrity (74).
Additional studies will be required to determine the mechanism(s) of action and pharmacological utility of protecting
mtDNA integrity in the many pulmonary disorders in which
acute oxidant stress plays a pathogenic role.
It has been appreciated for some time that ROS contribute to
the evolution of ALI/acute respiratory distress syndrome
(ARDS) and other lung diseases, possibly through an adverse
effect on mitochondrial function (11). Indeed, ⬃40 clinical
trials have examined the effect of antioxidant strategies in ALI
and multiple organ system failure, but the therapeutic effects of
antioxidant strategies have been unimpressive (88). These
disappointing outcomes may be attributed in part to the heterogeneous nature and onset of ALI/ARDS that tend to confound
design and interpretation of clinical trials. There also remains
considerable scientific uncertainty about molecular targets of
antioxidant drug action. For example, nonselective antioxi-
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sitive to oxidant stress (25), fails to display oxidative base
modifications in hypoxia (31). Perhaps this is because in
hypoxia, the matrix, where mtDNA resides, becomes more
reduced in hypoxia as ROS are vectored into the IMS and
cytosol (92).
In marked contrast to the lack of effect of hypoxia-induced
mitochondrial ROS generation on mtDNA integrity, when cells
or intact lung tissue are challenged with exogenous ROS the
nuclear genome is largely spared of lesions whereas mtDNA is
extensively damaged (7, 16, 32, 97). One question that emerges
from this interesting observation pertains to the biological
value in having a mitochondrial genome sensitive to damage
by exogenous oxidants whereas the nuclear genome is relatively resistant. A potential answer, discussed subsequently, is
that mtDNA functions as a “fuse” or a “sentinel molecule”
such that before an externally applied oxidant stress rises to a
level that threatens the nuclear genome with mutation, oxidative mtDNA damage triggers both death of the affected cell and
promotes the dissemination of signals to “alarm” neighboring
and itinerant cells.
Multiple lines of evidence support the idea that mtDNA
serves as a molecular sentinel dictating cell survival in response to oxidant stress. As just noted, in virtually all cell types
studied thus far mtDNA is highly sensitive to ROS-induced
damage (7, 32, 97). There is also a conspicuous association
between mtDNA damage and ROS-mediated cell death, with
the propensity for cytotoxicity inversely related to the efficiency of mtDNA repair (32, 37). Genetic modulation of the
first and rate-limiting step in mtDNA repair, mediated by Ogg1
(19), a DNA glycosylase that detects and excises oxidatively
damaged purines, coordinately regulates ROS- and reactive
nitrogen species (RNS)-induced mtDNA damage and cell
death in all cultured cell populations so far examined (14, 21,
47, 67, 72, 73). Particularly relevant here are studies using
mitochondrially targeted Ogg1 constructs, which eliminate the
possibility that the biological consequences of Ogg1 overexpression are related to effects on nuclear DNA repair. Studies
in pulmonary artery endothelial cells show that overexpression
of mitochondrially targeted Ogg1 reduces sensitivities to both
oxidative mtDNA damage and cytotoxicity evoked by exogenous ROS stressors (21, 77). Conversely, reduction in total cell
Ogg1 by using siRNA, although not sensitizing nuclear DNA
to oxidative damage, leads to persistence of ROS-induced
damage to the mitochondrial genome, increased apoptosis, and
cytotoxicity (78).
Although these observations suggest that mtDNA integrity is
a determinant of ROS-induced cytotoxicity, many questions
remain about the specific pathway(s) by which oxidative
mtDNA damage dictates lung cell survival and function. The
link between ROS-induced mtDNA damage and cell death
traditionally has been ascribed to the idea that persistent
damage leads to diminished mtDNA transcription, defective
ETC function and to resulting formation of a regenerative cycle
of excessive proapoptotic ROS production and increasing
mtDNA damage that ultimately culminates in mitochondrially
driven cell death (11, 39, 75). However, this traditional model
has never been thoroughly explored, and, more importantly,
emerging evidence suggests that other pathways are equally
plausible. Protective actions of mtDNA repair enzymes against
ROS-induced lung cell death and dysfunction might not be
linked to mtDNA repair, but rather to their interaction with
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dants may disrupt signaling required for physiological adaption, such as hypoxia-mediated ventilation-perfusion matching
in the injured lung, lung cell survival, and recovery. Strategies
currently available also may not target the key sentinel molecule(s) triggering adverse cellular effects of ROS. Perhaps, as
suggested by the foregoing discussion, mtDNA could be such
a sentinel molecule.
Mitochondrial DNA DAMP Release and Its Cellular Targets
in Lung Disease
ROS
D-loop
Bioenergetic
defect, cell death
and dysfunction
mtDNA
Damageinduced
mtDNA
Fragmentation
into DAMPs
Mitochondrial
Matrix
PTP
IMS
Cytoplasm
Cellautogenous
TLR9 activation
NALP3
Inflammasome
Activation
Endosome
Plasma Membrane
?
Extracellular Space
Local Inflammation,
TLR9-dependent Immune System Activation,
and Injury Propagation
Fig. 2. Fate and biological actions of the oxidatively damaged mitochondrial
genome that warrant characterization of mtDNA as a “sentinel molecule.” See
text for details. DAMPs, damage associated molecular patterns; PTP, permeability transition pore.
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An intriguing question to emerge from the prospect that
mtDNA damaged by oxidation in the setting of oxidant lung
injury pertains to the fate of the molecule. As has been
suggested from studies in other experimental systems (20), if
mtDNA damage is severe and not promptly repaired, does the
molecule fragment, after which it is exported from the organelle, possibly as part of mitophagy? And if oxidatively damaged mtDNA fragments are exported from mitochondria, what
are the next destinations of the fragments and do they play a
role in disease pathogenesis? These questions, depicted in Fig.
2, are discussed below.
If unrepaired, damaged mtDNA is rapidly degraded (81).
Interestingly, mtDNA so damaged displays a higher abundance
of sugar-phosphate backbone lesions and abasic sites relative
to oxidized base products, which would be expected to fragment the DNA rather then lead to persistent mutagenic lesions.
There is scant evidence concerning the fate of mtDNA fragments formed as a consequence of oxidative damage. In a
study using isolated mitochondria subjected to an exogenous
oxidant stress, Garcia and Chavez (27) reported that mtDNA
fragments less than 1,000 bp were lost from the organelle via
the permeability transition pore. Studies in macrophages also
indicate that the permeability transition pore is required for
cytosolic accumulation of mtDNA after immunological stimulation (61), but whether there is some specificity for fragment
size or other attributes is unknown. In cardiomyocytes subjected to hemodynamic stress-induced mitochondrial damage,
mtDNA fragments accumulate in autolysosomes where, on the
one hand, DNase II is capable of degrading the fragments to
biological insignificance (65). On the other hand, mtDNA
fragments escaping degradation appear capable of autonomously activating endosomal TLR9 (65). In immunologically
stimulated macrophages, mtDNA fragments accumulating in
the cytoplasm function as a coactivator in the NALP3-dependent activation of caspase 1 (61). Importantly, both TLR9 and
NALP3 activation can initiate downstream proinflammatory
signaling.
In severely injured (100) and critically ill human subjects
(62) mtDNA accumulates in the circulation, where it functions
as a DAMP and causes TLR9-dependent activation of the
innate immune system. Here, too, the mechanism by which
mtDNA fragments exit cells has received little study. In IL-5or ␥-IFN-primed eosinophils, lipopolysaccharide causes the
ROS-dependent accumulation of mtDNA fragments in the
extracellular space. The release process, described as “catapultlike,” occurred rapidly and was not dependent on death of the
cells (98). Whether a similar process accounts for release of
mtDNA from other types of oxidatively or mechanically
stressed cells is entirely unknown. Indeed, whether mtDNA
DAMP release is inextricably linked to autophagy, mitophagy,
or cell death remains to be established.
As noted previously, mtDNA is normally localized in a
multiprotein complex within the mitochondrial matrix, referred
to as a nucleoid, which contains the essential transcription
factor and structural protein TFAM (9, 30, 44). TFAM has a
high binding affinity for mtDNA and remains associated when
DNA is immunoprecipitated from lysed cells (43). TFAM is a
functional and structural homologue of the nuclear DNA binding protein HMGB1, and both TFAM and HMGB1 are potent
ligands for RAGE. In the context promoting inflammation,
RAGE ligands are shown to amplify the immune responses to
pattern-recognizing Toll-like receptors (43). In contrast to
nuclear DNA, which is nonimmunogenic, mtDNA, like viral
and bacterial DNA, is enriched with hypomethylated CpG
motifs, which are natural ligands for TLR9, a highly specific
intracellular (endosomal) pattern recognizing receptor (50).
Given these properties of TFAM and mtDNA, it is not surprising that TFAM remains associated with mtDNA following cell
necrosis, and this complex is particularly potent in terms of
promoting TLR9-responsive immune cell activation (43).
Whereas many cell types are capable of responding to
exogenous and endogenous danger signals, including neutro-
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A
TFAM
RAGE
mtDNA
IFNα
T-reg
TLR9
Cell Damage
+/Contamination
Tissue Repair
Antimicrobial
Defense
pDC
TNFα
MФ
T-eff
Local
Inflammation
Negative Feedback
PMN
6000
No Treatment
CpGA DNA - IFNα
TFAM + CpGA - IFNα
CpGA DNA - TNFα
TFAM + CpGA - TNFα
*
5000
*
4000
‡
3000
2000
†
‡
1000
†
s
0
pD
w
Lo
H
ig
h
pD
C
C
s
Supernatant Cytokine Concentration
(pg/ml)
B
Fig. 3. Plasmacytoid dendritic cells (pDCs) play a central role in orchestrating
both pro- and anti-inflammatory responses. A: during acute tissue injury
mitochondrial DNA (mtDNA) and mitochondrial transcription factor A
(TFAM) released from damaged cells and contaminating bacteria is sensed by
pDCs to promote simultaneous release of TNF-␣ and IFN-␣, which promotes
the simultaneous activation of proinflammatory and regulatory immune cells.
Thus pDCs are poised to orchestrate a localized and self-limited inflammatory
response. Presumably, excessive pDCs activation (e.g., massive cell injury)
could promote systemic pro- and anti-inflammatory immune responses. B: in
splenocyte cultures representing complex immune cell interactions typically
occurring in vivo release of IFN-␣ and TNF-␣ in response to immunogenic
(CpGA) DNA is enhanced by TFAM and depletion of pDCs [by magnetic
MicroBead selection (Miltenyi Biotec, Auburn, CA)], representing ⬍3% of the
total cell population from the splenocyte cultures, is shown to greatly suppress
the immune response. Thus pDCs are important “amplifiers” of the immune
response to TFAM and DNA. *P ⬍ 0.01, compared with corresponding CpGA
DNA treatment alone; †P ⬍ 0.01, relative to matching treatment in the High
pDC group; and ‡P ⬍ 0.01, compared with corresponding CpGA DNA
treatment alone and the matching treatment in the high-pDC group. PMN,
neutrophil; M⌽, macrophage; T-eff, effector CD4⫹ T cell; T-reg, regulatory
T cell.
lungs results in the release of Type 1 interferons, which are
capable of suppressing ALI by altering the balance between
proinflammatory (e.g., IL-1␤, IL-6) and anti-inflammatory
(e.g., IL-10) cytokines (5). Finally, lung DCs present antigen to
and thereby activate regulatory T cells (Tregs), which, in turn,
contribute to the resolution of inflammation during the subacute phase of ALI (1) (Fig. 3A). Thus the current evidence
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phils (PMNs), monocytes, and even epithelial cells (50), dendritic cells (DCs) are specialized for sensing and processing
immunogenic antigens to initiate and coordinate complex and
sustained immune responses. Despite being present in low
abundance (⬍1%) in blood and other tissues, immature DCs,
particularly plasmacytoid DCs (pDCs), and immature myeloid
DCs (iDCs) are uniquely equipped to initiate and ultimately
sustain sterile immune responses to immunogenic DNA, including viral and bacterial DNA. Immature DCs are unique in
their capacity to simultaneously promote pro- and anti-inflammatory immune responses. They are distinguished from other
immune cell lines by the lack of expression of common T cell,
B cell, or myeloid cell surface markers and are further discriminated by their ability to produce large quantities of type I
interferons in response to immunogenic (e.g., viral or bacterial)
RNA and DNA, recognized by TLR7/8 and TLR9, respectively. Activated iDCs and pDCs, through the production of
TNF-␣, further promote “bystander” activation of regional
macrophages, are induced to mature and are targeted to various
tissues via expression of specific chemokine receptors [e.g.,
CCR2 targets DCs to lung (53), where they are capable of
presenting antigen via MHC class II molecules to promote
potent T cell-mediated adaptive and counterregulatory immune
responses (Fig. 3)].
In the context of acute cell damage, immature DCs respond
specifically to mitochondrial danger signals; indeed, the
TFAM/mtDNA complex has been shown to be sufficient to
promote the simultaneous release of TNF-␣ and IFN-␣ from
purified pDCs (42, 43) (Fig. 3A). Moreover, using a splenocyte
culture model closely approximating the complex interaction
of immune cells in vivo, TFAM was found to augment the
immune response to CpGA DNA (a readily available surrogate
for mtDNA). Furthermore, the depletion of pDCs, representing
a small fraction of the total splenocyte culture population,
dramatically suppressed the innate immune response to TFAM/
CpG complex, demonstrating the capacity of pDCs to amplify
both proinflammatory and counterregulatory immune pathways (Fig. 3B). Together, these data indicate that TFAM
remains associated with mtDNA following acute cell damage,
that TFAM serves to augment the immune response to
mtDNA, and that immature DCs are important sensors for the
TFAM/mtDNA complex and important regulators of the sterile
immune response to these mitochondrial danger signals.
New evidence indicates that DCs, particularly pDC-like
macrophages, are of critical importance during the pathogenesis of ALI in the context of sterile inflammation, and mitochondrial danger signals are incriminated as proximal mediators. Immunogenic DNA, such as is present in bacteria and
mitochondria, is sufficient to promote ALI in animal models
(46, 87), and mtDNA has been specifically linked to the
pathogenesis of ALI in setting of sterile inflammation induced
experimentally by acid aspiration (17), surgical trauma (100),
and acute liver necrosis (55). PMNs, through activation of
TLR9, appear to play a major role in initiating ALI under these
conditions. By contrast, recruitment of pDC-like macrophages
to the lung is shown to attenuate ALI induced by LPS,
particularly in the acute phase of lung injury (49). These
pDC-like macrophages express CCR2 and are recruited to the
lung by CC chemokine ligand-2 (CCL2), whereupon they
facilitate the clearance of damaged cells (52). In addition to
enhanced clearance of damaged cells, pDC activation in the
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NEW LUNG BIOLOGY OF MITOCHONDRIA
suggests that the acute damage inflicted by activated PMNs is
counterbalanced by pDCs in the context of ALI induced by
mitochondrial danger signals.
Circulating Mitochondria DNA: A Biomarker of Sepsis and
ARDS in the ICU
Mitochondrial Biogenesis to the Rescue
Decades of laboratory and clinical studies have revealed that
acute systemic inflammatory syndromes, including sepsis, generate substantial macromolecular damage to mitochondria, particularly in the cells of highly metabolically active tissues like
the liver, heart, and kidneys (83, 85). Such mitochondrial
damage may be indiscernible in vivo because it does not
necessarily activate cell death pathways, and some of the same
factors that damage mitochondria, e.g., ROS, can also activate
the process of mitochondrial biogenesis (86). Recently mitochondrial biogenesis has been detected even in distal lung
cells, including alveolar type II epithelial cells (6). Indeed, all
cells that rely on aerobic respiration and oxidative phosphorylation for energy conservation express a robust genetic program of mitochondrial quality control that is designed to
maintain a fully functional complement of mitochondria
through the conjoined activities of mitochondrial biogenesis
and mitochondrial autophagy (mitophagy) (89). These closely
regulated processes serve a prosurvival capacity by allowing
cells to quickly replace metabolically dysfunctional mitochondria with fresh organelles from a pool of undamaged organelles
before energy failure (Fig. 4).
During inflammation mitochondrial damage is caused by the
overproduction of reactive oxygen and nitrogen species (ROS/
RNS) at multiple sites, leading to the inactivation of critical
mitochondrial macromolecules through biochemical oxidation,
nitration, and/or S-nitrosation. The critical mitochondrial targets are diverse, including many proteins, lipids, and mtDNA
that embody integral mitochondrial processes including protein
importation, transcription, protein synthesis, tricarboxylic acid
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As noted above, accumulating evidence suggests that mitochondrial damage may engender the export mtDNA DAMPs
that activate innate immune responses and possibly propagate
injury to distant targets. In this regard, although biomarkers can
be useful for identifying patients with critically illness or
assessing the responses to the therapies for the patients in the
intensive care unit (ICU), a small number of biomarkers
reflecting the disease severity are commonly measured in
clinical practice (e.g., lactate) (71). The question thus arises as
to whether mtDNA DAMP may be a useful biomarker.
Inflammasomes are multiprotein complexes that activate
caspase-1 and downstream immune responses, including the
maturation and secretion of proinflammatory cytokines (i.e.,
IL-1␤ and IL-18) (18). Recent studies show that inflammasomes are involved in the pathogenesis of various diseases
such as infection, cancer, and metabolic diseases (18). Inflammasomes are activated by a wide range of signals of pathogenic
and nonpathogenic origins (host cell derived molecules including extracellular ATP, hyaluronan, amyloid-␤ fibrils, and uric
acid crystals) (18).
To examine the involvement of inflammasome in critical
illnesses, Choi and colleagues (22, 23) first performed gene
expression profiling analysis of lungs from mice subjected to
mechanical ventilation (MV), an experimental animal model of
ALI. Specific changes in expression were identified for
caspase-activator domain-10, IL-18 receptor-1 (Il18r1), and
Il1r2 and IL-1␤ (Il1b) as genes following MV. Since genes
representing inflammasome complex molecules and the downstream cytokines were regulated in animal models of ALI, it
was then determined whether inflammasome family genes are
also regulated in human critical illnesses such as ARDS, a
lethal disease in ICU, and sepsis, a major cause of ARDS and
a leading cause of death in ICU. Total blood RNA was
extracted from patients admitted in medical ICU to determine
the global gene expression profile of ICU controls, patients
with systemic inflammatory response syndrome (SIRS), sepsis,
and sepsis/ARDS. The gene expression profiling analysis revealed that inflammasome-associated genes (e.g., asc and Il1b)
were upregulated in patients with sepsis/ARDS compared with
SIRS (23). In addition, plasma levels of IL-18, a representative
inflammasome-mediated proinflammatory cytokine, at the time
of medical ICU admission were higher with increasing disease
severity categories (control, SIRS, sepsis, and sepsis/ARDS)
and the highest in sepsis/ARDS patients (23). Importantly,
patients with elevated IL-18 levels at the time of medical ICU
hospitalization had increased mortality (23). In mice, MV
enhanced IL-18 levels in the serum and bronchoalveolar lavage
fluid (23). Furthermore, IL-18 neutralizing antibody treatment,
or genetic deletion of IL-18 or caspase-1, reduced lung injury
in response to MV (23). These data suggest critical roles of
inflammasome-mediated immune responses in ICU patients.
Recent studies suggest mtDNA is a potent inflammasome
activator (61) and is released from mitochondria to the cytosol
or the extracellular space as a mitochondrial DAMP. In addi-
tion, exogenous administration of disrupted mitochondria containing mtDNA induces acute inflammatory tissue injuries
(100). Therefore, we examined whether the levels of circulating mtDNA are associated with disease severity or mortality in
ICU (62). Circulating cell-free mtDNA level was higher in
ICU patients who died within 28 days of medical ICU admission as well as in patients with sepsis or ARDS in the ICU.
Medical ICU patients with an elevated mtDNA level had an
increase in their odds of dying within 28 days of ICU admission, whereas no evidence for association was noted in nonmedical ICU patients (e.g., surgical ICU patients). There was
no evidence that the association between mtDNA level and
medical ICU mortality was attenuated in models adjusted for
clinical covariates, including Acute Physiology and Chronic
Health Evaluation (APACHE) II score, sepsis, or ARDS. The
inclusion of an elevated mtDNA to a clinical model (including
age, gender, race/ethnicity, APACHE II score, and sepsis)
improved the net reclassification of 28-day mortality among
medical ICU patients. The improvement in net reclassification
remained significant even after additionally adjusting for measures of procalcitonin or lactate, commonly used biomarkers in
ICU. Similar relationships between circulating mtDNA and
outcome have been noted in patients with severe trauma (82) or
patients with sepsis in the emergency room (51). Thus circulating mtDNA could serve as a viable plasma biomarker in
medical ICU patients and may represent an important pathogenic determinant that contributes to a exaggerated systemic
inflammatory response observed in patients with sepsis or
ARDS.
Perspectives
NEW LUNG BIOLOGY OF MITOCHONDRIA
L969
cycle function, electron transport, heme synthesis, and control
of the mitochondrial permeability transition pore (69). The
upshot of damage from elevated ROS production is the compromised efficiency of oxidative phosphorylation, which may
destabilize energy homeostasis and lead to further inflammation that may perpetuate the mitochondrial damage (101).
However, the production of mitochondrial ROS also provides
signals to activate redox-sensitive transcriptional pathways that
regulate mitochondrial quality control and trigger the synthesis
of counterinflammatory mediators, such as IL-10 (70). Moreover, the enhanced induction of enzymatic NO and CO production also promotes the expression of a large group of genes
that is essential for mitochondrial biogenesis (63, 84).
A number of the transcriptional pathways activated by the
NO synthase 2 (NOS2) and heme oxygenase-1 (HO-1) enzymes for the induction of mitochondrial biogenesis during
inflammation have been worked out over the last several years.
For both enzymes, the endogenous gaseous product (NO or
CO) is directly involved in the induction mechanisms through
the activation of redox-sensitive pathways. For NOS2, as for
the other NOS isoforms, a key step involves the cyclic GMPdependent phosphorylation of cyclic AMP-responsive element-binding protein 1 (CREB1), which undergoes translocation to the nucleus to activate the gene for PGC-1a, a critical
coactivator of mitochondrial biogenesis and oxidative metabolism. Activated PGC-1a protein is a partner for nuclear
respiratory factor-1 (NRF-1) and NRF-2 (GABPA); these are
both essential transcription factors for mitochondrial biogenesis in most cells. For HO-1, heme catabolism releases CO,
which binds to the reduced terminal oxidase of the respiratory
chain and increases the mitochondrial H2O2 leak rate. The
increased H2O2 production activates several prosurvival kinases, such as Akt, as well as the transcription factor Nfe2l2
(Nrf2) (68). Nrf2, in its gene transactivation function, binds to
antioxidant response elements (ARE) in the NRF-1 gene promoter, whereas Akt phosphorylates GSK-3b, which relieves a
restriction on the entry of Nrf2 into the nucleus. Akt phosphorylates the NRF-1 protein as well, allowing both transcription
factors to gain entrance to the nucleus to induce an assemblage
of genes required for mitochondrial biogenesis.
At counterpoise to mitochondrial biogenesis is the removal
of senescent or damaged mitochondria that no longer perform
oxidative phosphorylation or are triggering oxidative damage
to nearby cellular structures. Such damaged mitochondria also
propagate certain danger signals, for example, inflammasome
activation (101). The deconstruction of obsolescent mitochondria by mitophagy, a form of selective macroautophagy, entails
the envelopment of the mitochondrial cargo within doublemembrane vesicles or autophagosomes, which fuse with lysosomes to degrade the cargo. The rate of mitophagy is governed
by the extent of mitochondrial damage, but also by the cell’s
energy requirement and its capacity to meet this mitochondrial
disposal function (99). Thus, from the cellular perspective,
optimal stress-stabilizing strategies match energy requirements
with the rates of mitophagy and mitochondrial biogenesis to
ensure energy sufficiency and mitochondrial quality control.
In the mammalian lung, mitochondrial energy provision is
an important function of many of the roughly 40 cell types
present there. In the parenchyma, for instance, alveolar type II
cells are enriched in mitochondria, which provide ATP to
support surfactant synthesis, secretion, and recycling. These
cells also initiate mitochondrial biogenesis during ALI, pneumonia, hyperoxia, and other stresses through induction of the
HO-1/CO and NOS2 systems. These alveolar type II cell
mitochondria also proliferate to support not only surfactant
metabolism, but multiple other energy-dependent functions in
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Fig. 4. Integrated process of mitochondrial quality control in the maintenance of mitochondrial homeostasis.
The diagram illustrates the simplified relationship between mitochondrial biogenesis and selective mitochondrial autophagy or mitophagy. The cycle is accelerated
by oxidative stress and is monitored and regulated by
redox sensors including the physiological gases.
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NEW LUNG BIOLOGY OF MITOCHONDRIA
Mitochondrial Transfer to the Rescue
The foregoing discussion raises the prospect that mitochondrial oxygen sensing, maintenance of mtDNA integrity to
prevent mtDNA fragmentation and release in the form of
mtDNA DAMPs, and mitochondrial biogenesis may be important determinants of outcome in sepsis. A related and desirable
goal of mitochondrial-directed therapy in severe illness is to
prevent mitochondrial dysfunction (11). Clearly, the attendant
decreased ATP production could causes failure of various
aspects of cell function that in combination could lead to
multiorgan failure in ALI/ARDS. The cause of mitochondrial
failure is not well understood, but one possibility is that NF-␬B
activation, occurring secondary to TLR4 ligation by bacterial
endotoxin, causes transcriptional upregulation of inducible
nitric oxide synthase. The increased nitric oxide thus produced
reacts with mitochondrial superoxide to cause peroxynitration,
hence dysfunction of mitochondrial electron transport proteins
(69). Although other mechanisms may also apply, the end
result is failure of cellular bioenergetics as well documented by
the reduction of tissue ATP levels. The therapeutic objective,
therefore, would be to replenish dysfunctional mitochondria of
host cells with fresh mitochondria.
The question is how does one replenish mitochondria? One
possibility is to give exogenous mitochondria with the expectation that cells will take up the mitochondria by endocytosis
and thereby repair the ATP deficit. However, the caveat here is
that free mitochondria in the extracellular space might act as
DAMPs that activate pattern recognition receptors that exacerbate tissue inflammation and injury (100).
The other option is to enable intercellular mitochondrial
transfer, a process that has been reported in cultured cells (76).
Islam et al. (40) now show that the process also occurs in vivo
and that mitochondrial transfer from exogenously administered
BMSCs (bone-marrow-derived stromal cells) to the alveolar
epithelium protects against LPS-induced ALI. A more recent
report by Ahmad et al. (2) also affirm that mitochondrial
transfer from BMSCs to the airway epithelium occurs in vivo
under inflammatory conditions. In this paper, the authors show
(1)
Fig. 5. Sequence of events leading to
mitochondrial transfer from alveolus-attached
bone-marrow-derived stromal cells (BMSCs)
to the alveolar epithelium. The sketch of the
distal airway (left) shows the arrival of airway instilled BMSCs (MSC) in alveoli composed of alveolar type 1 and type 2 cells (AT2
and AT2, respectively) and alveolar macrophages (AM). Subsequent processes in the
alveolus (right) are 1) BMSCs adhere to the
alveolar epithelium through Cx43 interactions; 2) gap junctions form between BMSCs
and the epithelium; 3) calcium levels increase
in the BMSCs; and 4) BMSCs release mitochondria-containing microvesicles which enter the epithelium and release their mitochondria in the cytosol.
(2)
AM
MSC
(3)
Cx43
AT2
AT1
(4)
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the transition to type I epithelium, such as those involved in
maintaining alveolar barrier integrity. In fact, mitochondrial
proliferation precedes the proliferation of type II cells as well
as their differentiation into type I epithelium. Under many of
these same conditions, as discussed elsewhere in this Perspectives article, autophagy programs, including mitophagy, are
activated to eliminate damaged proteins and other damaged
organelles from the cell.
In the future, our understanding of the basic intracellular
mechanisms of mitochondrial quality control in the lung may
be brought into line with newly emerging roles for mitochondrial depletion, immune cell scavenging of exported mitochondrial components, and adoptive mitochondrial rescue-by-transfer during acute and chronic lung injury. The importance of
these intricate extra- and intercellular processes will hinge on
the relative effectiveness of mitochondrial biogenesis and mitophagy under pathogenic conditions of varying type, intensity,
and severity that are currently being investigated in different
experimental settings.
Perspectives
NEW LUNG BIOLOGY OF MITOCHONDRIA
GRANTS
The authors’ research described in this Perspectives article was supported
by grants from the National Institutes of Health (R01 HL35440 and R01
HL122062 to P. T. Schumacker, R01 HL058234 and R0 1 HL113614 to M. N.
Gillespie, R01 HL055330 and P01 HL114501 to A. M. K. Choi, R01 AI62740
and R01 AI083912 to E. D. Crouser, P01 HL108801 and R01 AI095424 C. A.
Piantadosi, and RO1 HL36024 and RO1 HL57556 to J. Bhattacharya).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
P.T.S., M.N.G., E.D.C., and C.A.P. prepared figures; P.T.S., M.N.G., K.N.,
A.M.C., E.D.C., C.A.P., and J.B. drafted manuscript; P.T.S., M.N.G., K.N.,
A.M.C., E.D.C., C.A.P., and J.B. edited and revised manuscript; P.T.S.,
M.N.G., K.N., A.M.C., E.D.C., C.A.P., and J.B. approved final version of
manuscript.
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that the mitochondrial transfer abrogated airway inflammation
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