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Articles in PresS. Am J Physiol Lung Cell Mol Physiol (April 18, 2014). doi:10.1152/ajplung.00073.2014 1 2 L-00073-2014 Revised (clean) 3 Mitochondria in Lung Biology and Pathology: 4 More than just a powerhouse 5 6 7 8 9 Paul T. Schumacker1, Mark N. Gillespie2†, 10 Kiichi Nakahira3, Augustine M.K. Choi3, Elliott D. Crouser4 11 Claude A. Piantadosi5, and Jahar Bhattacharya6 12 13 14 1 Northwestern University Feinberg School of Medicine, Department of 15 Pediatrics, Chicago, IL, 2University of South Alabama College of Medicine, 16 Department of Pharmacology, Mobile, AL, 3Weill Cornell Medical College, 17 Department of Medicine, New York, NY, 4The Ohio State University College 18 of Medicine, Department of Internal Medicine, Columbus, OH, 19 University School of Medicine, Department of Medicine, Durham, NC, and 20 6 21 Cellular Biophysics, New York, NY Duke Columbia University Medical Center, Department of Physiology and 22 23 24 25 26 27 28 29 30 5 † Correspondence: e-mail: telephone: Mark N. Gillespie, Ph.D Department of Pharmacology College of Medicine University of South Alabama Mobile, AL 36688 [email protected] (251) 460-6497 Copyright © 2014 by the American Physiological Society. 31 32 ABSTRACT 33 An explosion of new information about mitochondria reveals that their importance 34 extends well beyond their time honored function as the “powerhouse of the cell.” In this 35 Perspective, we summarize new evidence showing that mitochondria are at the center 36 of a reactive oxygen species (ROS)-dependent pathway governing the response to 37 hypoxia and to mitochondrial quality control. The potential role of the mitochondrial 38 genome as a sentinel molecule governing cytotoxic responses of lung cells to ROS 39 stress also is highlighted. Additional attention is devoted to the fate of damaged mtDNA 40 relative to its involvement as a Damage Associated Molecular Pattern driving adverse 41 lung and systemic cell responses in severe illness or trauma. 42 strategies for replenishing normal populations of mitochondria after damage, either 43 through promotion of mitochondrial biogenesis or via mitochondrial transfer, are 44 discussed. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 Finally, emerging 61 62 63 INTRODUCTION “The powerhouse of the cell.” All of the contributors to the 2013 ATS 64 symposium forming the basis of this Perspective Article, and perhaps many of the 65 attendees, too, recalled this as their first introduction to mitochondria and often their first 66 memorable phrase in biology. Though still true, it now seems almost quaint given the 67 explosion of information about mitochondrial functions unrelated to energy metabolism 68 that has occurred over the past two decades or so. Our symposium was motivated 69 partly by this “explosion” and partly by the equally rapid accumulation of evidence that 70 mitochondria likely play crucial roles in lung disease with significant potential to serve as 71 targets in pulmonary and critical care medicine. 72 This Perspective article does not seek to comprehensively review all 73 developments in the mitochondrial field that bear on lung disease. Rather, it highlights 74 evolving concepts of how mitochondria contribute to normal lung health, and how 75 mitochondria could be targeted for specific intervention in lung disease. Having 76 registered this, we present concise summaries pertaining to mitochondria, reactive 77 oxygen species (ROS) and oxygen sensing in the lung, the role of the mitochondrial 78 genome as a sentinel molecule governing lung cell survival in response to oxidant 79 stress, the nature of mtDNA Damage Associated Molecular Patterns (DAMPs) 80 originating from the lung and their potential use and importance as biomarkers and 81 proinflammatory alarm signals, respectively. Finally, we explore the intriguing notion 82 that therapeutic strategies aimed at reconstituting normal mitochondrial population and 83 function may comprise therapeutic approaches in acute lung injury. 3 84 85 Mitochondrial oxygen sensing Mitochondria consume O2 at cytochrome c oxidase, the terminal complex in the 86 electron transport chain (ETC). Electron flux along the ETC is linked to proton 87 translocation across the inner mitochondrial membrane, resulting in the generation of an 88 electrochemical gradient that is used by the ATP synthase to generate ATP during 89 oxidative phosphorylation. As mitochondria convert O2 to water the intracellular O2 90 tension decreases, thereby generating a gradient for O2 diffusion into the cell from the 91 extracellular space. Mitochondria are the principal site of oxygen consumption in the 92 cell, so their oxygen tension is lower than any other organelle. This characteristic has 93 led investigators to ask whether mitochondria function as oxygen sensors in the cell 94 (64). A conceptually simple idea is that decreases in cellular O2 levels (tissue hypoxia) 95 would limit mitochondrial oxygen consumption, leading to redox changes as the ETC 96 complexes became saturated with electrons due to the loss of cytochrome c oxidase 97 activity (56). 98 systems that feed electrons into the ETC, such as the Krebs cycle. 99 conceptual problem with this idea arose from experimental studies showing that 100 mitochondria do not become limited by oxygen availability until the cell is nearly anoxic 101 (12, 95). Specifically, how could the mitochondria respond to physiological levels of 102 hypoxia if their ability to signal via redox alterations was not activated until all of the 103 oxygen was gone? For many years this paradox represented a conceptual barrier to 104 the idea that mitochondria might be involved in oxygen sensing. Some investigators 105 suggested that specialized oxygen sensors such as the glomus cells of the carotid body 106 might circumvent this problem by expressing special isoforms of cytochrome c oxidase These redox changes would then be reflected in the redox status of 4 However, a 107 with low apparent affinity for O2 (59). In those cells, electron transport and oxygen 108 consumption would become O2 supply-limited at milder levels of hypoxia, allowing the 109 mitochondria to detect subtle decreases in cellular oxygenation (58). 110 experimental evidence for the existence of such a low-affinity oxidase has been 111 reported. Moreover, mammalian cells with high-affinity forms of cytochrome c oxidase 112 are still capable of detecting and responding to hypoxia. However, no 113 A second hypothesized model of oxygen sensing was promoted by Michelakis, 114 Archer and Weir, who sought to explain how pulmonary artery smooth muscle cells 115 detect hypoxia and trigger acute hypoxic pulmonary vasoconstriction (4). Their model 116 proposed the existence of an intracellular oxidase that would generate more ROS at 117 high O2 levels and less under hypoxic conditions. According to that model, ROS would 118 then activate voltage-dependent potassium (Kv) channels in the plasma membrane. 119 Conversely, the lesser oxidation of these channels during hypoxia would promote 120 channel closure, causing membrane depolarization that would lead to the entry of 121 extracellular Ca2+ through L-type calcium channels. However, when they tested the 122 effect of gp91-(phox) deletion on this response, using mice with genetic deletion of 123 NOX2, they found that the cellular response to hypoxia was still present (3). They 124 subsequently considered that mitochondria were the source of these ROS signals, 125 based on studies using various mitochondrial inhibitors (57). However, a conceptual 126 problem with that model has been the consistent failure of diverse chemical antioxidants 127 or the expression of enzymatic antioxidant systems to activate hypoxic responses in a 128 normoxic cell. 5 129 A third model of mitochondrial oxygen sensing (FIGURE 1) arose from initial 130 studies showing that intracellular ROS levels actually increase during hypoxia (13, 26). 131 Parallel studies using chemical mitochondrial inhibitors implicated complex III as the 132 likely source of ROS generation. 133 because it seemed counter-intuitive, and was based on the use of non-specific 134 fluorescent probes to detect intracellular ROS levels and pharmacological inhibitors to 135 block the ETC (34, 90). 136 inhibitors at various concentrations reported conflicting responses. To address this, 137 Waypa et al. utilized a novel genetically encoded fluorescent protein sensor to detect 138 intracellular ROS levels. The initial sensor, HSP-FRET, exhibited ratiometric behavior 139 that obviated an important limitation of the fluorescent chemical probes (91). 140 Specifically, ratiometric sensors provide a signal that is independent of the light intensity 141 used to excite the probe. 142 pulmonary artery smooth muscle cells confirmed the previous observation that ROS 143 levels increase in the cytosol during hypoxia (91). 144 during hypoxia was shown to be required for the increase in cytosolic Ca2+ activation in 145 pulmonary artery smooth muscle cells. Further advances in this hypoxia-induced ROS 146 signaling model came with the introduction of roGFP, a redox-sensitive mutant of green 147 fluorescent protein (24, 35). This ratiometric sensor provides a specific measure of 148 protein thiol oxidation/reduction status in the cell. Two important features contributed to 149 the importance of this advance. First, its oxidation is reversible in the cell allowing it to 150 be calibrated, analogous to the behavior of ratiometric Ca2+ sensors. 151 expression can be targeted to specific intracellular compartments, allowing assessment This paradoxical response was initially criticized Importantly, different laboratory groups using the same Importantly, studies using HSP-FRET expressed in 6 Moreover, the increase in ROS Second, its 152 of redox at the subcellular level. Using roGFP expressed in cytosol, mitochondrial 153 intermembrane space and in the mitochondrial matrix, Waypa et al. found that 154 significant differences in basal oxidant status exist across these compartments in 155 PASMC, with the cytosol existing in a more reduced state, the mitochondrial matrix in a 156 highly oxidized state, and the intermembrane space at an intermediate level (92). 157 During acute hypoxia, oxidation status in the intermembrane space and the cytosol 158 increased, whereas oxidation in the mitochondrial matrix decreased. These findings 159 were consistent with a model in which the release of superoxide from complex III to the 160 intermembrane space increases during hypoxia, allowing the ROS to potentially exit to 161 the cytosol and initiate redox signaling involved in triggering the release of intracellular 162 calcium stores (94). By contrast, hypoxia caused an attenuation of oxidant stress in 163 mitochondrial matrix. 164 compartments could reflect a distinct operation of two independent mechanisms 165 controlling ROS generation during hypoxia in the two domains. 166 possibility that ROS alters the direction of ROS secretion from the membrane – toward 167 the matrix during normoxia and toward the intermembrane space during hypoxia – has 168 not been ruled out. The opposing redox responses to hypoxia in these two Alternatively, the 169 In either case, studies were needed to more critically test the hypothesis that the 170 mitochondrial electron transport chain is the source of ROS signaling during hypoxia. 171 Accordingly, Waypa et al. developed a genetic model by generating a mouse to permit 172 conditional deletion of the Rieske iron-sulfur protein (RISP) (93). This nuclear encoded 173 protein is critical for the function of complex III, where it accepts an electron from 174 ubiquinol, the carrier that moves electron into complex III from complexes I and II (34). 7 175 ROS generation from complex III can occur after RISP accepts an electron from 176 ubiquinol, generating the intermediate free radical, ubisemiquinone. 177 electron on ubisemiquinone is normally transferred to the b-cytochromes in the 178 complex, but can potentially be transferred instead to O2, generating superoxide. If 179 RISP is deleted, ubisemiquinone, and thus superoxide, cannot be generated at complex 180 III (33, 54). Accordingly, that group studied PASMC isolated from the conditional RISP 181 mice and used Cre recombinase expression to delete the RISP gene. After loss of 182 RISP was confirmed, subsequent roGFP studies demonstrated that the hypoxia- 183 induced changes in redox status in the cytosol, intermembrane space and the 184 mitochondrial matrix were abolished. 185 functional complex III was required for the changes in redox observed in different 186 subcellular compartments during hypoxia (93). In parallel studies, it was found that the 187 transient increase in cytosolic Ca2+ in PASMC during acute hypoxia was abrogated in 188 cells where RISP had been deleted (93). 189 The remaining These studies therefore demonstrated that a A technical limitation of studies using cultured PASMC is the possibility that the 190 cells will undergo a phenotypic shift in culture. 191 cannot provide an assessment of the role of those responses in the intact lung. 192 Accordingly, that group conducted in vivo studies in which RISP was deleted from 193 smooth muscle cells by breeding the conditional RISP mice with a transgenic line 194 expressing a smooth muscle-specific, tamoxifen-inducible Cre (96). The resulting mice 195 were administered tamoxifen to activate nuclear localization of the Cre in smooth 196 muscle cells, and subsequent deletion of RISP from smooth muscle was confirmed. 197 The acute pulmonary vasoconstriction response was then compared in the knockout 8 Moreover, studies in cultured cells 198 and control mice, by measuring the right ventricular systolic pressure (RVSP) response 199 to acute alveolar hypoxia. Control mice exhibited a significant increase in RVSP during 200 5% O2 ventilation, which was significantly attenuated in the RISP-deficient mice (93). 201 These findings indicate that a functional complex III is required for the acute 202 vasopressor response to hypoxia in the lung. 203 Does the same mitochondrial ROS signal trigger other functional responses to 204 hypoxia? Mungai et al. tested the hypothesis that hypoxia activates the cellular energy 205 sensor, AMP-dependent kinase (AMPK), through an ROS-dependent mechanism. In 206 PASMC, they found that ROS generation during moderate hypoxia led to AMPK 207 activation, even in the absence of any increase in AMP or ADP concentrations (60). 208 Those findings suggest that mitochondrial ROS signals activate AMPK as an early 209 response to hypoxia, allowing it to activate protective mechanisms before the cell 210 progresses into a bioenergetic crisis where AMP and ADP concentrations are 211 increased. 212 Do specific antioxidants block these responses? To test this, Sabharwal et al. 213 reasoned that the expression of an enzymatic scavenger of H2O2 exclusively in the 214 mitochondrial intermembrane space (IMS) should abolish functional responses to 215 hypoxia by preventing ROS released from complex III to reach the cytosol (79). 216 Peroxiredoxin-5 (PRDX5) is an atypical 2-cysteine-containing peroxidase that is 217 normally expressed in multiple subcellular compartments including the mitochondrial 218 matrix (45). This enzyme functions as a monomer, whereas other H2O2 scavengers 219 only function as multimers. They targeted its expression to the intermembrane space 220 using the N-terminal sequence that directs the SMAC/Diablo protein to that 9 221 compartment (66), and confirmed correct localization using immunogold electron 222 microscopy (79). 223 PASMC, it attenuated the increase in ROS in the IMS and the cytosol during hypoxia. 224 Importantly, the PRDX5-expressing cells also showed a significant attenuation of the 225 acute hypoxia-induced activation of cytosolic Ca2+. 226 independent demonstration that ROS release from the mitochondrial inner membrane to 227 the IMS and subsequently to the cytosol is critical for triggering acute responses to 228 hypoxia in PASMC. When PRDX5 was expressed in the intermembrane space of These findings provide an 229 Previous studies by this group had shown that the stabilization of Hypoxia- 230 Inducible Factor-1alpha (HIF-1) is triggered by hypoxia-induced release of ROS from 231 mitochondria. In PASMC, hypoxia also triggers stabilization of HIF- 232 prolyl hydroxylase, which targets the protein for degradation by hydroxylating conserved 233 proline residues during normoxia (38, 41). 234 PRDX5 in the IMS, hypoxia-induced HIF-1 stabilization was inhibited in a dose- 235 dependent manner (79). In PASMC expressing graded levels of 236 Collectively, these findings indicate that mitochondria function as more than 237 simple factories supplying ATP to the cell. Through the regulated release of ROS to the 238 cytosol, these organelles have the capacity to orchestrate a diverse group of adaptive 239 responses to hypoxia. In that regard, the moniker of “cellular oxygen sensors” clearly 240 applies to these organelles. 241 242 10 243 Sentinel functions of mitochondrial DNA (mtDNA) in pulmonary 244 vascular cells 245 The nuclear and mitochondrial genomes differ in certain fundamental respects. 246 For example, in comparison to the nuclear genome, the mitochondrial genome is tiny. 247 Even if all the mtDNA molecules in cells with high mitochondrial densities are totaled, it 248 is still a small fraction of the size of the nuclear genome. Following from this, mtDNA 249 encodes for only a handful of proteins compared to the many thousands encoded by 250 nuclear DNA. Second, the structure and regulation of the mitochondrial genome is 251 comparatively simple. Unlike nuclear DNA, in which the state of compaction, its 252 interactions with multiple transcription factors and co-activators, and epigenetic 253 determinants are all exquisitely regulated processes culminating in precise control of 254 gene expression, the mitochondrial genome is expressed as a single unit and its 255 transcription is controlled by a handful of mtDNA binding proteins, particularly 256 Mitochondrial Transcription Factor A (TFAM, (10, 44). Instead of compaction within a 257 nucleosome context, a single molecule of mtDNA is associated with about 30 core 258 proteins in a structure known as the nucleoid. Most if not all of the enzymes needed for 259 mtDNA replication and mtRNA expression appear to be components of the nucleoid (9). 260 Another difference between mitochondrial and nuclear DNA particularly relevant 261 to the present discussion is that mtDNA is about 50-fold more sensitive to oxidative 262 damage (7, 16, 32, 97). Reasons for this heightened sensitivity are not entirely known, 263 but have been attributed to a comparatively simple process of mtDNA repair 264 (mitochondria seem to contain only base excision repair while the nucleus has multiple, 11 265 overlapping pathways (8), and the prospect that the open structure of mtDNA is more 266 accessible to DNA-damaging ROS. In light of the forgoing comments that mitochondrial 267 ROS generation plays a central role in hypoxic signal transduction, the question can be 268 raised as to whether mtDNA is damaged by oxidation in hypoxic environments. While 269 DNA integrity across the entire mitochondrial genome in hypoxic cells has not been 270 examined, a large expanse of the mtDNA coding region - including the “common 271 deletion sequence” known to be sensitive to oxidant stress (25) - fails to display 272 oxidative base modifications in hypoxia (31). Perhaps this is because in hypoxia, the 273 matrix, where mtDNA resides, becomes more reduced in hypoxia as ROS are vectored 274 into the intermembrane space and cytosol (92). 275 In marked contrast to the lack of effect of hypoxia-induced mitochondrial ROS 276 generation on mtDNA integrity, when cells or intact lung tissue are challenged with 277 exogenous ROS the nuclear genome is largely spared of lesions while mtDNA is 278 extensively damaged (7, 16, 32, 97). One question that emerges from this interesting 279 observation pertains to the biological value in having a mitochondrial genome sensitive 280 to damage by exogenous oxidants while the nuclear genome is relatively resistant 281 potential answer, discussed subsequently, is that mtDNA functions as a “fuse” or a 282 “sentinel molecule” such that before an externally applied oxidant stress rises to a level 283 that threatens the nuclear genome with mutation, oxidative mtDNA damage triggers 284 both death of the affected cell and promotes the dissemination signals to “alarm” 285 neighboring and itinerant cells. A 286 Multiple lines of evidence support the idea that mtDNA serves as a molecular 287 sentinel dictating cell survival in response to oxidant stress. As just noted, in virtually all 12 288 cell types studied thus far mtDNA is highly sensitive to ROS-induced damage (7, 32, 289 97). 290 mediated cell death, with the propensity for cytotoxicity inversely related to the efficiency 291 of mtDNA repair (32, 37). Genetic modulation of the first and rate-limiting step in mtDNA 292 repair, mediated by Ogg1 (19), a DNA glycosylase that detects and excises oxidatively 293 damaged purines – coordinately regulates ROS- and reactive nitrogen species-induced 294 mtDNA damage and cell death in all cultured cell populations so far examined (14, 21, 295 47, 67, 72, 73). Particularly relevant here are studies using mitochondrially-targeted 296 Ogg1 constructs, which eliminate the possibility that the biological consequences of 297 Ogg1 over-expression are related to effects on nuclear DNA repair. 298 pulmonary artery endothelial cells show that over-expression of mitochondrially-targeted 299 Ogg1 reduces sensitivities to both oxidative mtDNA damage and cytotoxicity evoked by 300 exogenous ROS stressors (21, 77). 301 siRNA, while not sensitizing nuclear DNA to oxidative damage, leads to persistence of 302 ROS-induced damage to the mitochondrial genome, increased apoptosis and 303 cytotoxicity (78). There is also a conspicuous association between mtDNA damage and ROS- Studies in Conversely, reduction in total cell Ogg1 using 304 While these observations suggest that mtDNA integrity is a determinant of ROS- 305 induced cytotoxicity, many questions remain about the specific pathway(s) by which 306 oxidative mtDNA damage dictates lung cell survival and function. The link between 307 ROS-induced mtDNA damage and cell death traditionally has been ascribed to the idea 308 that persistent damage leads to diminished mtDNA transcription, defective ETC 309 function, and resulting formation of a regenerative cycle of excessive pro-apoptotic ROS 310 production and increasing mtDNA damage that ultimately culminates in mitochondrially- 13 311 driven cell death (11, 39, 75). However, this traditional model has never been 312 thoroughly explored, and more importantly, emerging evidence suggests that other 313 pathways are equally plausible. Protective actions of mtDNA repair enzymes against 314 ROS-induced lung cell death and dysfunction might not be linked to mtDNA repair, but 315 rather, to their interaction with oxidatively damaged mtDNA or mtDNA damage-related 316 proteins. 317 defective Ogg1 mutant protected against asbestos-induced apoptosis in A549 cells (67). 318 Because the repair-defective enzyme, like the wild-type enzyme, bound aconitase, the 319 possibility was advanced that Ogg1 influences ROS-mediated signaling events 320 downstream of mtDNA damage (80). In a related context, it has recently been shown 321 that Ogg1 in complex with free 8-oxoguanine serves as a guanine exchange factor to 322 activate Ras-mediated signaling (29), but whether this occurs in intact cells with ROS- 323 induced mtDNA damage has not been established. In this regard, over-expression of a mitochondrially-targeted DNA repair- 324 As a means of exploring involvement of oxidative mtDNA damage in intact lung 325 and animal models of pulmonary disease, Wilson and colleagues developed novel 326 fusion-protein constructs targeting DNA repair glycosylases, either Ogg1 or the bacterial 327 enzyme Endo III, to mitochondria (48). Early work showed that these fusion protein 328 constructs prevented both hydrogen peroxide-induced mtDNA damage and increases in 329 the vascular filtration coefficient in isolated perfused rat lungs (16). The Ogg1 construct 330 also attenuated ventilator-induced lung injury and mortality in mice (36) and hyperoxia- 331 induced dysmorphogenesis in fetal rat lung explants (28). 332 suggest that mitochondrially-targeted Ogg1 inhibits mortality and acute lung injury 333 evoked by intratracheal P. aeruginosa in rats (15). 14 Preliminary studies also 334 These observations on the effectiveness of increasing mtDNA repair in isolated 335 lungs and animal models are a step towards determining if mtDNA integrity is a 336 legitimate pharmacologic target in lung disease. However, there are many unresolved 337 issues. As noted above, some pertain to the specific mechanism whereby increased 338 mtDNA repair protects against cytotoxicity. As an extension of this, whereas protection 339 of mtDNA integrity in cultured cells inhibits ROS-induced cytotoxicity, the involvement of 340 cytotoxicity per se in intact animal models of acute lung injury is complex. Taking this 341 analysis one step further, it is easy to envision how oxidative mtDNA damage could, by 342 different mechanisms, mediate different types of cellular responses in lung disease. For 343 example, ROS-induced cytotoxicity, driven by persistent mtDNA damage, defective 344 mtDNA transcription and attendant bioenergetic insufficiency, could be lethal to some 345 populations of lung cells, while oxidant-induced alterations in mtDNA-protein stability 346 might acutely influence behavior of other lung cell populations secondary to alterations 347 in cytoskeletal integrity (74). 348 mechanism(s) of action and pharmacologic utility of protecting mtDNA integrity in the 349 many pulmonary disorders where acute oxidant stress plays a pathogenic role. Additional studies will be required to determine the 350 It has been appreciated for some time that ROS contribute to the evolution of 351 ALI/ARDS and other lung diseases, possibly through an adverse effect on mitochondrial 352 function (11). Indeed, about 40 clinical trials have examined the effect of anti-oxidant 353 strategies in acute lung injury and multiple organ system failure, but the therapeutic 354 effects of anti-oxidant strategies have been unimpressive (88). These disappointing 355 outcomes may be attributed in part to the heterogeneous nature and onset of ALI/ARDS 356 that tend to confound design and interpretation of clinical trials. There also remains 15 357 considerable scientific uncertainty about molecular targets of anti-oxidant drug action. 358 For example, non-selective antioxidants may disrupt signaling required for physiologic 359 adaption, such as hypoxia-mediated ventilation-perfusion matching in the injured lung, 360 lung cell survival and recovery. Strategies currently available also may not target the 361 key sentinel molecule(s) triggering adverse cellular effects of ROS. 362 suggested by the foregoing discussion, mtDNA could be such a sentinel molecule. Perhaps, as 363 364 Mitochondrial DNA DAMP Release and its Cellular Targets in Lung 365 Disease 366 An intriguing question to emerge from the prospect that mtDNA damaged by 367 oxidation in the setting of oxidant lung injury pertains to the fate of the molecule. As has 368 been suggested from studies in other experimental systems (20), if mtDNA damage is 369 severe and not promptly repaired, does the molecule fragment after which it is exported 370 from the organelle, possibly as part of mitophagy? And if oxidatively-damaged mtDNA 371 fragments are exported from mitochondria, what are the next destinations of the 372 fragments and do they play a role in disease pathogenesis? These questions, depicted 373 in FIGURE 2, are discussed below. 374 If unrepaired, damaged mtDNA is rapidly degraded (81). Interestingly, mtDNA so 375 damaged displays a higher abundance of sugar-phosphate backbone lesions and 376 abasic sites relative to oxidized base products, which would be expected to fragment 377 the DNA rather then lead to persistent mutagenic lesions. There is scant evidence 378 concerning the fate of mtDNA fragments formed as a consequence of oxidative 16 379 damage. In a study using isolated mitochondria subjected to an exogenous oxidant 380 stress, Garcia and Chavez reported that mtDNA fragments less than 1000bp were lost 381 from the organelle via the permeability transition pore (27). Studies in macrophages 382 also indicate that the permeability transition pore is required for cytosolic accumulation 383 of mtDNA after immunologic stimulation (61), but whether there is some specificity for 384 fragment size or other attributes is unknown. In cardiomyocytes subjected to 385 hemodymamic stress-induced mitochondrial damage, mtDNA fragments accumulate in 386 autolysosomes where, on the one hand, Dnase II is capable of degrading the fragments 387 to biological insignificance (65). 388 degradation appear capable of autonomously activating endosomal TLR9 (65). In 389 immunologically-stimulated macrophages, mtDNA fragments accumulating in the 390 cytoplasm function as a co-activator in the NALP3-dependent activation of caspase 1 391 (61). Importantly, both TLR9 and NALP3 activation can initiate downstream pro- 392 inflammatory signaling. On the other hand, mtDNA fragments escaping 393 In severely injured (100) and critically ill human subjects (62) mtDNA 394 accumulates in the circulation where it functions as a DAMP and causes TLR9- 395 dependent activation of the innate immune system. Here, too, the mechanism by which 396 mtDNA fragments exit cells has received little study. 397 interferon-primed 398 accumulation of mtDNA fragments in the extracellular space. The release process – 399 described as “catapult-like” – occurred rapidly and was not dependent on death of the 400 cells (98). Whether a similar process accounts for release of mtDNA from other types of 401 oxidatively- or mechanically-stressed cells is entirely unknown. Indeed, whether mtDNA eosinophils, lipopolysaccharide 17 In interleukin-5- or gamma causes the ROS-dependent 402 DAMP release is inextricably linked to autophagy, mitophagy or cell death remains to be 403 established. 404 As noted previously, mtDNA is normally localized in a multi-protein complex 405 within the mitochondrial matrix, referred to as a nucleoid, which contains the essential 406 transcription factor and structural protein TFAM (9, 30, 44). TFAM has a high binding 407 affinity for mtDNA and remains associated when DNA is immunoprecipitated from lysed 408 cells (43). TFAM is a functional and structural homologue of the nuclear DNA binding 409 protein HMGB1, and both TFAM and HMGB1 are potent ligands for RAGE. 410 context promoting inflammation, RAGE ligands are shown to amplify the immune 411 responses to pattern-recognizing Toll-like receptors (43). In contrast to nuclear DNA 412 which is non-immunogenic, mtDNA, like viral and bacterial DNA, is enriched with 413 hypomethylated CpG motifs, which are natural ligands for TLR9, a highly specific 414 intracellular (endosomal) pattern recognizing receptor (50). 415 TFAM and mtDNA, it is not surprising that TFAM remains associated with mtDNA 416 following cell necrosis, and this complex is particularly potent in terms of promoting 417 TLR9-responsive immune cell activation (43). In the Given these properties of 418 Whereas many cell types are capable of responding to exogenous and 419 endogenous danger signals, including PMNs, monocytes, and even epithelial cells (50), 420 dendritic cells are specialized for sensing and processing immunogenic antigens to 421 initiate and coordinate complex and sustained immune responses. 422 present in low abundance (<1%) in blood and other tissues, immature dendritic cells, 423 particularly plasmacytoid DCs (pDCs) and immature myeloid DCs (iDCs), are uniquely 424 equipped to initiate and ultimately sustain sterile immune responses to immunogenic 18 Despite being 425 DNA, including viral and bacterial DNA. Immature DCs are unique in their capacity to 426 simultaneously promote pro- and anti-inflammatory immune responses. 427 distinguished from other immune cell lines by the lack of expression of common T-cell, 428 B-cell or myeloid cell surface markers, and are further discriminated by their ability to 429 produce large quantities of type I interferons in response to immunogenic (e.g., viral or 430 bacterial) RNA and DNA, recognized by TLR7/8 and TLR9, respectively. 431 iDCs and pDCs, through the production of TNFa, further promote “bystander” activation 432 of regional macrophages, are induced to mature, and are targeted to various tissues via 433 expression of specific chemokine receptors [e.g., CCR2 targets DCs to lung (53), where 434 they are capable of presenting antigen via MHC class II molecules to promote potent T 435 cell-mediated adaptive and counter-regulatory immune responses (FIGURE 3). They are Activated 436 In the context of acute cell damage, immature DCs respond specifically to 437 mitochondrial danger signals; indeed, the TFAM/mtDNA complex has been shown to be 438 sufficient to promote the simultaneous release of TNFa and IFNa from purified pDCs 439 (42, 43) (FIGURE 3A). 440 approximating the complex interaction of immune cells in vivo, TFAM was found to 441 augment the immune response to CpGA DNA (a readily available surrogate for mtDNA). 442 Furthermore, the depletion of pDCs, representing a small fraction of the total splenocyte 443 culture population, dramatically suppressed the innate immune response to TFAM/CpG 444 complex, demonstrating the capacity of pDCs to amplify both proinflammatory and 445 counter-regulatory immune pathways (FIGURE 3B). Together, these data indicate that 446 TFAM remains associated with mtDNA following acute cell damage, that TFAM serves 447 to augment the immune response to mtDNA, and immature DCs are important sensors Moreover, using a splenocyte culture model closely 19 448 for TFAM/mtDNA complex and important regulators of the sterile immune response to 449 these mitochondrial danger signals. 450 New evidence indicates that DCs, particularly pDC-like macrophages, are of 451 critical importance during the pathogenesis of acute lung injury in the context of sterile 452 inflammation, and mitochondrial danger signals are incriminated as proximal mediators. 453 Immunogenic DNA, such as is present in bacteria and mitochondria, is sufficient to 454 promote ALI in animal models (46, 87), and mtDNA has been specifically linked to the 455 pathogenesis of ALI in setting of sterile inflammation induced experimentally by acid 456 aspiration(17), surgical trauma (100), and acute liver necrosis (55). 457 through activation of TLR9, appear to play a major role in initiating ALI under these 458 conditions. By contrast, recruitment of pDC-like macrophages to the lung is shown to 459 attenuate ALI induced by LPS, particularly in the acute phase of lung injury (49). These 460 pDC-like macrophages express CCR2 and are recruited to the lung by CC chemokine 461 ligand-2 (CCL2), whereupon they facilitate the clearance of damaged cells (52). In 462 addition to enhanced clearance of damaged cells, pDC activation in the lungs results in 463 the release of Type 1 interferons which are capable of suppressing ALI by altering the 464 balance between pro-inflammatory (e.g. IL-1b, IL-6) and anti-inflammatory (e.g., IL-10) 465 cytokines (5). Finally, lung DCs present antigen to and thereby activate regulatory T 466 cells (Tregs), which, in turn, contribute to the resolution of inflammation during the sub- 467 acute phase of ALI(1) (FIGURE 3A). 468 acute damage inflicted by activated PMNs is counterbalanced by pDCs in the context of 469 ALI induced by mitochondrial danger signals. Neutrophils, Thus, the current evidence suggests that the 470 20 471 Circulating Mitochondria DNA: A Biomarker of Sepsis and ARDS in 472 the ICU 473 As noted above, accumulating evidence suggests that mitochondrial damage 474 may engender the export mtDNA DAMPs that activate innate immune responses and 475 possibly propagate injury to distant targets. In this regard, while biomarkers can be 476 useful for identifying patients with critically illness or assessing the responses to the 477 therapies for the patients in the intensive care unit (ICU), a small number of biomarkers 478 reflecting the disease severity are commonly measured in clinical practice (e.g. lactate, 479 (71). The question thus arises as to whether mtDNA DAMP may be a useful biomarker. 480 Inflammasomes are multi-protein complexes that activate caspase-1 and 481 downstream immune responses, including the maturation and secretion of pro- 482 inflammatory cytokines (i.e. interleukin (IL)-1β and IL-18) (18). Recent studies show 483 inflammasomes are involved in the pathogenesis of various diseases such as infection, 484 cancer and metabolic diseases (18). Inflammasomes are activated by a wide range of 485 signals of pathogenic and non-pathogenic origins (host cell derived molecules including 486 extracellular ATP, hyaluronan, amyloid-β fibrils and uric acid crystals) (18). 487 To examine the involvement of inflammasome in critical illnesses, Choi and 488 colleagues first performed gene expression profiling analysis of lungs from mice 489 subjected to mechanical ventilation (MV), an experimental animal model of acute lung 490 injury (ALI). Specific changes in expression were identified for caspase-activator 491 domain-10, interleukin-18 receptor-1 (Il18r1), and Il1r2 and interleukin-1 (Il1b) as 492 genes following MV (22, 23). Since genes representing inflammasome complex 21 493 molecules and the downstream cytokines were regulated in animal models of ALI, it was 494 then determined whether inflammasome family genes are also regulated in human 495 critical illnesses such as acute respiratory distress syndrome (ARDS), a lethal disease 496 in ICU, and sepsis, a major cause of ARDS and a leading cause of death in ICU. Total 497 blood RNA was extracted from patients admitted in medical ICU to determine the global 498 gene expression profile of ICU controls, patients with systemic inflammatory response 499 syndrome (SIRS), sepsis, and sepsis/ARDS. The gene expression profiling analysis 500 revealed that inflammasome-associated genes (e.g. asc and Il1b) were up-regulated in 501 patients with sepsis/ARDS when compared to SIRS (23). In addition, plasma levels of 502 IL-18, a representative inflammasome-mediated pro-inflammatory cytokine, at the time 503 of medical ICU admission were higher with increasing disease severity categories 504 (control, SIRS, sepsis and sepsis/ARDS) and the highest in sepsis/ARDS patients (23). 505 Importantly, patients with elevated IL-18 levels at the time of medical ICU hospitalization 506 had increased mortality (23). In mice, MV enhanced IL-18 levels in the serum and 507 bronchoalveolar lavage fluid (23). Furthermore, IL-18 neutralizing antibody treatment, or 508 genetic deletion of IL-18 or caspase-1, reduced lung injury in response to MV (23). 509 These data suggest critical roles of inflammasome-mediated immune responses in ICU 510 patients. 511 Recent studies suggest mtDNA is a potent inflammasome activator (61) and is 512 released from mitochondria to the cytosol or the extracellular space as a mitochondrial 513 DAMP. In addition, exogenous administration of disrupted mitochondria containing 514 mtDNA induces acute inflammatory tissue injuries (100). 515 whether the levels of circulating mtDNA are associated with disease severity or mortality 22 Therefore, we examined 516 in ICU (62). Circulating cell-free mtDNA level was higher in ICU patients who died within 517 28 days of medical ICU admission as well as in patients with sepsis or ARDS in the 518 ICU. Medical ICU patients with an elevated mtDNA level had an increase in their odds 519 of dying within 28 days of ICU admission, while no evidence for association was noted 520 in non-medical ICU patients (e.g. surgical ICU patients). There was no evidence that the 521 association between mtDNA level and medical ICU mortality was attenuated in models 522 adjusted for clinical covariates, including Acute Physiology and Chronic Health 523 Evaluation (APACHE) II score, sepsis, or ARDS. The inclusion of an elevated mtDNA to 524 a clinical model (including age, gender, race/ethnicity, APACHE II score, and sepsis) 525 improved the net-reclassification of 28-day mortality among medical ICU patients. The 526 improvement in net reclassification remained significant even after additionally adjusting 527 for measures of procalcitonin or lactate, commonly used biomarkers in ICU. Similar 528 relationships between circulating mtDNA and outcome have been noted in patients with 529 severe trauma (82) or patients with sepsis in the emergency room (51). 530 circulating mtDNA could serve as a viable plasma biomarker in medical ICU patients 531 and, may represent an important pathogenic determinant that contributes to a 532 exaggerated systemic inflammatory response observed in patients with sepsis or 533 ARDS. Thus, 534 535 Mitochondrial biogenesis to the rescue 536 Decades of laboratory and clinical studies have revealed that acute systemic 537 inflammatory syndromes, including sepsis, generate substantial macromolecular 23 538 damage to mitochondria, particularly in the cells of highly metabolically active tissues 539 like the liver, heart, and kidneys (83, 85). Such mitochondrial damage may be 540 indiscernible in vivo because it does not necessarily activate cell death pathways, and 541 some of the same factors that damage mitochondria – e.g., ROS - can also activate the 542 process of mitochondrial biogenesis (86). Recently mitochondrial biogenesis has even 543 been detected even in distal lung cells, including alveolar type II epithelial cells(6). 544 Indeed, all cells that rely on aerobic respiration and oxidative phosphorylation for energy 545 conservation express a robust genetic program of mitochondrial quality control that is 546 designed to maintain a fully functional complement of mitochondria through the 547 conjoined 548 (mitophagy) (89). These closely regulated processes serve a pro-survival capacity by 549 allowing cells to quickly replace metabolically dysfunctional mitochondria with fresh 550 organelles from a pool of undamaged organelles before energy failure (FIGURE 4). activities of mitochondrial biogenesis and mitochondrial autophagy 551 During inflammation mitochondrial damage is caused by the over-production of 552 reactive oxygen and nitrogen species (ROS/RNS) at multiple sites, leading to the 553 inactivation of critical mitochondrial macromolecules through biochemical oxidation, 554 nitration, and/or S-nitrosation. The critical mitochondrial targets are diverse, including 555 many proteins, lipids, and mtDNA that embody integral mitochondrial processes 556 including protein importation, transcription, protein synthesis, TCA cycle function, 557 electron transport, heme synthesis, and control of the mitochondrial permeability 558 transition pore (69). The upshot of damage from elevated ROS production is the 559 compromised efficiency of oxidative phosphorylation, which may destabilize energy 560 homeostasis, and lead to further inflammation that may perpetuate the mitochondrial 24 561 damage (101). However, the production of mitochondrial ROS also provides signals to 562 activate redox-sensitive transcriptional pathways that regulate mitochondrial quality 563 control and trigger the synthesis of counter-inflammatory mediators, such as IL-10 (70). 564 Moreover, the enhanced induction of enzymatic NO and CO production also promotes 565 the expression of a large group of genes that is essential for mitochondrial biogenesis 566 (63, 84). 567 A number of the transcriptional pathways activated by the NO synthase 2 (NOS2) 568 and heme oxygenase-1 (HO-1) enzymes for the induction of mitochondrial biogenesis 569 during inflammation have been worked out over the last several years. For both 570 enzymes, the endogenous gaseous product (NO or CO) is directly involved in the 571 induction mechanisms through the activation of redox-sensitive pathways. For NOS2, 572 as for the other NOS isoforms, a key step involves the cyclic GMP-dependent 573 phosphorylation of cyclic AMP-responsive element-binding protein 1 (CREB1), which 574 undergoes translocation to the nucleus to activate the gene for PGC-1a, a critical co- 575 activator of mitochondrial biogenesis and oxidative metabolism. Activated PGC-1a 576 protein is a partner for nuclear respiratory factor-1 (NRF-1) and NRF-2 (GABPA); these 577 are both essential transcription factors for mitochondrial biogenesis in most cells. For 578 HO-1, heme catabolism releases CO, which binds to the reduced terminal oxidase of 579 the respiratory chain and increases the mitochondrial H2O2 leak rate. The increased 580 H2O2 production activates several pro-survival kinases, such as Akt, as well as the 581 transcription factor Nfe2l2 (Nrf2)(68). Nrf2, in its gene transactivation function, binds to 582 anti-oxidant response elements (ARE) in the NRF-1 gene promoter, while Akt 583 phosphorylates GSK-3b, which relieves a restriction on the entry of Nrf2 into the 25 584 nucleus. Akt phosphorylates the NRF-1 protein as well, allowing both transcription 585 factors to gain entrance to the nucleus to induce an assemblage of genes required for 586 mitochondrial biogenesis. 587 At counterpoise to mitochondrial biogenesis is the removal of senescent or 588 damaged mitochondria that no longer perform oxidative phosphorylation or are 589 triggering oxidative damage to nearby cellular structures. Such damaged mitochondria 590 also propagate certain danger signals, for example, inflammasome activation (101). The 591 deconstruction of obsolescent mitochondria by mitophagy, a form of selective 592 macroautophagy, entails the envelopment of the mitochondrial cargo within double- 593 membrane vesicles or autophagosomes, which fuse with lysosomes to degrade the 594 cargo. The rate of mitophagy is governed by the extent of mitochondrial damage, but 595 also by the cell’s energy requirement and its capacity to meet this mitochondrial 596 disposal function (99). Thus, from the cellular perspective, optimal stress-stabilizing 597 strategies match energy requirements with the rates of mitophagy and mitochondrial 598 biogenesis in order to ensure energy sufficiency and mitochondrial quality control. 599 In the mammalian lung, mitochondrial energy provision is an important function of 600 many of the roughly 40 cell types present there. In the parenchyma for instance, 601 alveolar type II cells are enriched in mitochondria, which provide ATP to support 602 surfactant synthesis, secretion, and recycling. These cells also initiate mitochondrial 603 biogenesis during acute lung injury, pneumonia, hyperoxia, and other stresses through 604 induction of the HO-1/CO and NOS2 systems. These alveolar type II cell mitochondria 605 also proliferate to support not only surfactant metabolism, but multiple other energy- 606 dependent functions in the transition to type I epithelium, such as those involved in 26 607 maintaining alveolar barrier integrity. In fact, mitochondrial proliferation precedes the 608 proliferation of type II cells as well as their differentiation into type I epithelium. Under 609 many of these same conditions, as discussed elsewhere in this Perspective, autophagy 610 programs, including mitophagy, are activated in order to eliminate damaged proteins 611 and other damaged organelles from the cell. 612 In the future, our understanding of the basic intracellular mechanisms of 613 mitochondrial quality control in the lung will be brought into line with newly emerging 614 roles for mitochondrial depletion, immune cell scavenging of exported mitochondrial 615 components, and adoptive mitochondrial rescue-by-transfer during acute and chronic 616 lung injury. The importance of these intricate extra- and intercellular processes will 617 hinge on the relative effectiveness of mitochondrial biogenesis and mitophagy under 618 pathogenic conditions of varying type, intensity, and severity that are currently being 619 investigated in different experimental settings. 620 621 Mitochondrial transfer to the rescue 622 The foregoing discussion raises the prospect that mitochondrial oxygen sensing, 623 maintenance of mtDNA integrity to prevent mtDNA fragmentation and release in the 624 form of mtDNA DAMPs, and mitochondrial biogenesis may be important determinants of 625 outcome in sepsis. 626 severe illness is to prevent mitochondrial dysfunction (11). 627 decreased ATP production could causes failure of various aspects of cell function that in 628 combination could lead to multi-organ failure in ALI/ARDS. The cause of mitochondrial A related and desirable goal of mitochondrial-directed therapy in 27 Clearly, the attendant 629 failure is not well understood, but one possibility is that NFκB activation, occurring 630 secondary to TLR4 ligation by bacterial endotoxin, causes transcriptional up regulation 631 of inducible nitric oxide synthase. The increased nitric oxide thus produced reacts with 632 mitochondrial superoxide to cause peroxynitration, hence dysfunction of mitochondrial 633 electron transport proteins (69). Although other mechanisms may also apply, the end 634 result is failure of cellular bioenergetics as well-documented by the reduction of tissue 635 ATP levels. The therapeutic objective therefore, would be to replenish dysfunctional 636 mitochondria of host cells with fresh mitochondria. 637 The question is how does one replenish mitochondria? One possibility is to give 638 exogenous mitochondria with the expectation that cells will take up the mitochondria by 639 endocytosis and thereby repair the ATP deficit. However the caveat here is that free 640 mitochondria in the extracellular space might act as DAMPs (damage-associated 641 molecular patterns) that activate pattern recognition receptors that exacerbate tissue 642 inflammation and injury (100). 643 The other option is to enable intercellular mitochondrial transfer, a process that 644 has been reported in cultured cells (76). Islam et al. now show that the process also 645 occurs in vivo and that mitochondrial transfer from exogenously administered BMSCs 646 (bone-marrow-derived stromal cells) to the alveolar epithelium protects against 647 lipopolysaccharide (LPS)-induced ALI (40). A more recent report by Ahmad et al. also 648 affirms that mitochondrial transfer from BMSCs to the airway epithelium occurs in vivo 649 under inflammatory conditions (2). 650 mitochondrial transfer abrogated airway inflammation and protected against bronchial 651 injury caused by mitochondrial dysfunction. These findings attest to the feasibility of In this paper, the authors show that the 28 652 mitochondrial transfer between specific cell types in vivo, indicating that it might be 653 applicable as a therapeutic tool in that mitochondrial transfer might correct the deficit of 654 mitochondrial function in affected cells through donation of fresh mitochondria from 655 BMSCs. 656 Islam et al. noted two important features regarding mitochondrial transfer. First, 657 the transfer did not occur when BMSCs were instilled in control lungs not exposed to 658 LPS, indicating that pathologic stress to the alveolar epithelium was essential. Second, 659 stabilization of BMSCs on the alveolar epithelium was also essential. The stabilization 660 occurred through formation of gap junctional channels (GJCs) between the BMSCs and 661 the alveolar epithelium. Knockdown of connexin 43, a GJC protein, blocked the BMSC 662 stabilization and also diminished the survival benefit accruing from BMSC instillation. 663 These findings indicate that for mitochondrial transfer to occur from a donor to a 664 recipient cell, a pre-conditioning of the interacting cell surfaces is critical. 665 In the study by Islam et al. the mechanism of mitochondrial transfer in alveoli 666 occurred through a sequence in which BMSCs released mitochondria-containing 667 nanotubes and microvesicles, and then the alveolar epithelium engulfed the 668 microvesicles, internalizing the mitochondria (FIGURE 5). 669 Miro1, a mitochondrial Rho-GTPase, is critical for this process since BMSCs lacking 670 Miro1 fail to transfer mitochondria to the airway epithelium. The mechanisms that 671 regulate microvesicle engulfment and release of the internalized mitochondria in the 672 alveolar cytosol are not clear. 673 internalized mitochondria were functional, since progressive increases of ATP levels 674 were detectable in the mitochondria-recipient epithelium. Islam et al showed that Ahmad et al. show that However, in Islam and coworkers found that the 29 675 endotoxin-induced mouse mortality decreased markedly following instillation of BMSCs 676 carrying normal mitochondria, but not when the BMSC mitochondria were made 677 dysfunctional by knockdown of the electron-transporting Riske iron-sulfur protein that 678 determines mitochondrial ATP production. Although mitochondrial transfer has been 679 shown in vitro and in feral dogs (62), the reports by Islam et al. and Ahmad et al indicate 680 that the process occurs in vivo to promote tissue repair in organ systems undergoing 681 pathologic stress. The applicability of mitochondrial transfer as a viable therapeutic 682 modality in inflammatory lung disease requires further consideration. 683 684 685 Acknowledgements 686 The authors’ research described in this Perspective was supported by grants from the National 687 Institutes of Health (R01 HL35440 and R01 HL122062 to P.T.S., R01 HL058234 and R0 1 688 HL113614 to M.N.G., R01 HL055330 and P01 HL114501 to A.M.K.C., R01 AI62740 and R01 689 AI083912 to E.D.C. P01 HL108801 and R01 AI095424 C.A.P., and RO1 HL36024 and RO1 690 HL57556 to J.B.) 691 30 692 References 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 1. Aggarwal NR, D'Alessio FR, Tsushima K, Sidhaye VK, Cheadle C, Grigoryev DN, Barnes KC, and King LS. Regulatory T cell-mediated resolution of lung injury: identification of potential target genes via expression profiling. 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III: Mitochondrial complex III. 1058 FIGURE 2: Fate and biological actions of the oxidatively damaged mitochondrial 1059 genome that warrant characterization of mtDNA as a “sentinel molecule”. See 1060 text for details. Abbreviations: DAMPs, Damage Associated Molecular Patterns; IMS, 1061 intermembrane space; PTP, permeability transition pore 1062 FIGURE 3: Plasmacytoid dendritic cells (pDCs) play a central role in orchestrating 1063 both pro- and anti-inflammatory responses. 1064 mitochondrial DNA 1065 contaminating bacteria is sensed by pDCs to promote simultaneous release of TNFα 1066 and IFNα, which promotes the simultaneous activation of proinflammatory and 1067 regulatory immune cells. Thus, pDCs are poised to orchestrate a localized, and self- 1068 limited inflammtory response. Presumably, excessive pDCs activation (e.g., massive 1069 cell injury) could promote systemic pro- and anti-inflammatory immune responses. 1070 Panel B: In splenocyte cultures representing complex immune cell interactions typically 1071 occurring in vivo release of IFNα and TNFα in response to immunogenic (CpGA) DNA is 1072 enhanced by TFAM, and depletion of pDCs [by magnetic MicroBead selection (Miltenyi 1073 Biotec, Auburn, CA)], representing <3% of the total cell populationfrom the splenocyte 1074 cultures, is shown to greatly suppress the immune response. Thus, pDCs are important 1075 “amplifiers” of the immune response to TFAM and DNA. (*p < 0.01, compared to Conceptual model of oxygen sensing by mitochondria through the (mtDNA) and TFAM 39 Panel A: During acute tissue injury released from damaged cells and 1076 corresponding CpGA DNA treatment alone, †p < 0.01; relative to matching treatment in 1077 the High pDC group, and ‡p < 0.01, compared to corresponding CpGA DNA treatment 1078 alone and the matching treatment in the High pDC group.) PMN = neutrophil; MФ = 1079 macrophage; T-eff = effector CD4+ T cell; T-reg = regulatory T cell 1080 FIGURE 4: 1081 maintenance of mitochondrial homeostasis. The diagram illustrates the simplified 1082 relationship between mitochondrial biogenesis and selective mitochondrial autophagy or 1083 mitophagy. The cycle is accelerated by oxidative stress and is monitored and regulated 1084 by redox sensors including the physiological gases. 1085 FIGURE 5: Sequence of events leading to mitochondrial transfer from alveolus- 1086 attached BMSCs to the alveolar epithelium. The sketch of the distal airway (LEFT) 1087 shows the arrival of airway instilled BMSCs (MSC) in alveoli composed of alveolar type 1088 1 and type 2 cells (AT2 and AT2, respectively) and alveolar macrophages (AM). 1089 Subsequent processes in the alveolus (RIGHT) are: (1) BMSCs adhere to the alveolar 1090 epithelium through Cx43 interactions; (2) Gap junctions form between BMSCs and the 1091 epithelium; (3) Calcium levels increase in the BMSCs; and (4) BMSCs release 1092 mitochondria-containing microvesicles which enter the epithelium and release their 1093 mitochondria in the cytosol. The integrated process of mitochondrial quality control in the 1094 1095 1096 1097 40 1098 1099 41 FIGURE 1 FIGURE 2 ROS D-loop Bioenergetic defect, cell death and dysfunction mtDNA Damageinduced mtDNA Fragmentation into DAMPs Mitochondrial Matrix IMS PTP Cytoplasm Cellautogenous TLR9 activation NALP3 Inflammasome Activation Endosome Plasma Membrane ? Extracellular Space Local Inflammation, TLR9-dependent Immune System Activation, and Injury Propagation FIGURE 3 A. TFAM RAGE mtDNA IFNα T reg T-reg TLR9 Cell Damage +/Contamination Tissue Repair Antimicrobial Defense pDC TNFα MФ T-eff Local Inflammation Negative Feedback * 6000 5000 No Treatment CpGA DNA - IFNα TFAM + CpGA - IFNα CpGA DNA - TNFα TFAM + CpGA - TNFα * 4000 ‡ 3000 † 2000 † 1000 ‡ pD w Lo pD Cs Cs 0 Hi gh (p g /m l) B. S u p e rn a ta n t C yto k in e C o n c e n tra tio n PMN FIGURE 4 FIGURE 5 (1) (2) AM MSC (3) Cx43 AT2 AT1 (4)