Mitochondrial dysfunction and the inflammatory response

The .pdf version of this research can be found at

1. Introduction
Inflammation, a basic biological response against invading pathogens, contributes to repairing damage and preventing further tissue or cell injury (Medzhitov, 2008). However, when the inflammatory response cannot be appropriately attenuated, multi-organ failure or chronic inflammation can develop (Medzhitov, 2008).

In this sense, increasing levels of pro-inflammatory cytokines have been associated with disease evolution and unfavorable outcomes in several pathological scenarios, such as chronic heart failure, peritoneal dialysis, pancreatitis, and cancer (Berres et al., 2011; Gravante et al., 2009; Hiller et al., 2011; Nevado et al., 2006; Yndestad et al., 2012).

While each of these disorders has a specific pathogenesis and disease evolution, they all involve inflammatory processes. In addition, the inflammatory process also has a role in mediating the aging process via changes in redox status and oxidative stress-induced inflammatory responses (Gilca et al., 2007); this process has been called inflammaging (Salminen et al., 2012). In fact, aging and low-grade inflammation involves many of the same molecular pathways, including oxidative stress and DNA damage pathways.

The molecular inflammation hypothesis of aging postulates that chronic low-grade inflammation in aging causes cumulative damage to biological macromolecules through the formation of oxygen radicals (Chung et al., 2009).

This process would eventually render cells more susceptible to damage and more sensitive to catabolism, processes associated with an exacerbated response to age-related diseases (Chung et al., 2009; Jian et al., 2011; Loeser, 2009). Hence, the risk of degenerative diseases and poor outcomes of acute processes rise exponentially with age.

This is also supported by studies showing that healthy centenarians exhibit low levels of inflammatory biomarkers (Franceschi, 2007).

Altered mitochondrial function is linked to several acute and chronic inflammatory diseases (Naik and Dixit, 2011). Additionally, mitochondria have long been proposed to play a key role in aging (Green et al., 2011).

As a consequence of their central role in ATP formation via the mitochondrial respiratory chain (MRC), mitochondria are the major source of reactive oxygen species (ROS) and are thus highly involved in oxidative stress processes (Brookes et al., 2004). However, mitochondria are also targets of these molecules (Brookes et al., 2004).

Under physiological conditions, about 1–3% of molecular oxygen is incompletely reduced during redox reactions in the MRC, and this in turn leads to production of the ROS superoxide anion (O2−) as a by-product of these reactions. In this scenario, complex interactions in antioxidant defense systems repress oxidative stress within mitochondria (Kienhöfer et al., 2009).

However, under pathological conditions, an excess of O2− ions are produced, which causes the activation of redox-sensitive transcription factors, such as the key regulator of tissue inflammation, nuclear factor-κB (NF-κB), and a subsequent increase in the expression of cytokines, chemokines, eicosanoids, inducible nitric oxide synthase (iNOS), and adhesion molecules (Chung et al., 2009).

In addition to alteration of signaling pathways regulated by ROS, the excess of ROS also drives oxidation of membrane lipids, proteins, and mitochondrial DNA (mtDNA) (Henze and Martin, 2003).

In particular, mtDNA seems to be especially sensitive to oxidative damage, and therefore susceptible to mutations, because of its close proximity to the site of ROS production and the lack of protective histones. In fact, aged individuals, whose cells have accumulated a high level of oxidative damage, exhibit an increased rate of mutagenesis in their mtDNA (Kujoth et al., 2005).

Moreover, superoxide anions can combine with nitric oxide (NO) to produce peroxynitrite (ONOO-), a powerful oxidant capable of affecting mitochondrial integrity (Brown, 2003; Escames et al., 2011).

Mitochondrial dysfunction drives mitochondrial genome mutagenesis, affecting genes encoding respiratory chain complexes and compromising the efficiency of oxidative phosphorylation (OXPHOS), which may lead to further mtDNA mutations and even mutations in the nuclear genome, thereby causing increased cell damage (Escames et al., 2011).

In this review, we aim to highlight the significance of mitochondrial dysfunction and to discuss the contribution of mitochondria to the development of inflammatory human diseases and the aging process.

2. Mitochondrial dysfunction may modulate inflammatory processes

It is well known that mitochondria are not only the primary source of ATP, but also participate in many other cell signaling events, such as regulation of calcium (Ca2 +) homeostasis, orchestration of apoptosis, and production of ROS, as mentioned above (Brookes et al., 2004).

In this sense, ROS are the major host defense mechanism against infection and harmful agents (Chen et al., 2012), and mitochondria are the principal mediators of inflammation (Tschopp, 2011).

During viral infection the pattern recognition receptors RIG-I and MDA5 attach to viral RNA allowing its interaction with a mitochondrial polypeptide adaptor, MAVS, which finally drives the production of type I interferon (Saitoh and Akira, 2010).

Interestingly, pathogens have recently been shown to have developed their own reciprocal defense systems; for example, Leishmania infection has been shown to cause marked upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation, potentially preventing ROS-mediated inactivation to suppress macrophage defense mechanisms and facilitate parasite survival (Basu Ball et al., 2011).

However, under dysregulated processes, when mitochondria are compromised by damage or mutations, excess O2− ions are produced, and cellular stress cannot be effectively resolved. Thus, mitochondria are the principal arbitrators of the pro-inflammatory status; they act through modulating innate immunity via redox-sensitive inflammatory pathways or direct activation of the inflammasome, a group of protein complexes whose activation results in immediate activation of caspase-1, thereby allowing for cleavage and subsequent activation of the inactive precursors IL-1β and IL-18 (Strowig et al., 2012).

Additionally, both of these pathways, i.e., the redox-sensitive inflammatory pathway and the inflammasome pathway, may work together to activate inflammatory cytokines, leading to an overstimulation of the inflammatory response (Escames et al., 2011).

Taken together, these recent studies have suggested the unexpected supplementary role of mitochondria as drivers of the inflammatory process.

2.1. Activation of redox-sensitive inflammatory pathways by mitochondrial impairment
Cellular systems that protect against oxidants involve antioxidative defense enzymes (superoxide dismutase [SOD], glutathione peroxidase [GPx], and catalase) (Kienhöfer et al., 2009), oxidant scavengers (vitamin E, vitamin C, carotenoids, uric acid, and polyphenols) and mechanisms to repair oxidant-induced damage to lipids, proteins, or DNA (Karger et al., 2012; Ugarte et al., 2010).

Despite these protective mechanisms, uncontrolled generation of ROS can overwhelm the capacity of cellular antioxidant protection causing mitochondrial dysfunction. In this sense, in vivo studies in transgenic mice showed that the overexpression of catalase targeted to mitochondria reduces age-associated pathologies and the mice have an extension in lifespan (Schriner et al., 2005).

Additionally, several studies have demonstrated that mitochondrial dysfunction may generate low-grade inflammatory and matrix degradation responses in several cell types via mitochondrial Ca2 + exchange, ROS generation, and NF-κB activation (Amma et al., 2005; Cillero-Pastor et al., 2008; Ichimura et al., 2003).

Thus, the flow of mitochondrial Ca2 + plays a crucial role in the evolution of cell signals and in the regulation of mitochondrial activity. Moreover, excessive mitochondrial Ca2+ accumulation has been shown to be critical for disease development (Dada and Sznajder, 2011; Tanaka et al., 2004).

For example, increased mitochondrial Ca2+ levels in cardiomyocytes contribute to cardiac inflammation and dysfunction after burn injury or sepsis (Maass et al., 2005).

Additionally, when cells are treated with inhibitors of mitochondrial Ca2 + exchange, ROS levels decrease significantly, leading to subsequent reductions in the levels of pro-inflammatory mediators. The rise in mitochondrial Ca2 +, a consequence of importing cytosolic Ca2 +, augments the mitochondrial production of ROS by different mechanisms, including 1) Ca2+ stimulation of the tricarboxylic acid cycle, which would enhance electron flow into the respiratory chain; 2) Ca2 + stimulation of NO synthase, elevating NO levels, which would in turn inhibit respiration at complex IV and enhance ROS generation; and 3) Ca2+ binding to cardiolipin (Brookes et al., 2004).

Thus, ROS production by mitochondrial electron transport could represent an intermediate step in pro-inflammatory gene expression/NF-κB signaling.
Mitochondrial dysfunction has also been shown to increase the responsiveness of several types of cells to cytokine-induced inflammatory responses, resulting in a significant amplification of the inflammatory response through ROS generation and NF-κB activation (Vaamonde-García et al., 2012; Nakahira et al., 2011).

When mitochondrial dysfunction was induced in normal synovial fibroblasts by an inhibitor of mitochondrial ATP synthase, oligomycin, an otherwise less-efficient concentration of IL-1β was as effective as a 10-times greater concentration of IL-1β would be in the absence of pretreatment with the mitochondrial inhibitor (Valcárcel-Ares et al., 2010).

In lung epithelial cells, pre-existing mitochondrial dysfunction induced by antisense oligonucleotides to complex III increased mitochondrial ROS generation, resulting in marked potentiation of ragweed pollen extract-induced accumulation of inflammatory cells in the airways (Aguilera-Aguirre et al., 2009).

In other cell types, mitochondrial impairment also increased the generation of ROS, resulting in potentiation of cytotoxicity or inflammatory cell accumulation (Bulua et al., 2011; Schulze-Osthoff et al., 1993).

Moreover, several studies have reported enhanced sensitivity to NF-κB activation in the context of mitochondrial dysfunction, suggesting that NF-κB binding sites may be essential for inflammatory gene expression (Ungvari et al., 2007; Vaamonde-García et al., 2012).

As expected, this inflammatory response is modulated by antioxidants. Additionally, these in vitro studies are supported by in vivo results. Thus, the effects of in vivo NF-kB inhibition (with pyrrolidinedithiocarbamate, PDTC or resveratrol) in aged rats significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness in vessels (Ungvari et al., 2007).

2.2. Mitochondrial modulation of the NLRP3 inflammasome.
Recent studies have shown mitochondria as important players in NLRP3 inflammasome activation (Green et al., 2011; Kepp et al., 2011; Nakahira et al., 2011; Shimada et al., 2012; Zhou et al., 2011).

Cell damage can initiate the accumulation of damage-associated molecular patterns (DAMPs), molecules recognized by the innate immune system and able to develop an inflammatory response (Krysko et al., 2011b) through the framework of the inflammasome and subsequent caspase-1 activation and pro-inflammatory cytokines release. Mitochondria have been recently identified as key sources of DAMPs (mito-DAMPs) playing a role in DAMP-modulated inflammation in different disorders, such as systemic inflammatory response syndrome (SIRS), rheumatoid arthritis (RA), cirrhosis, cancer, and heart diseases (Green et al., 2011; Zhang et al., 2010; Zitvogel et al., 2012), as well as the aging process (Salminen et al., 2012).

DAMPs activate the same receptors that detect pathogen-associated molecular patterns (PAMPs) (Krysko et al., 2011), such as Toll-like membrane receptors (TLRs) and cytoplasmic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs).

One of the best-studied NLRs, because of its solid association with several inflammatory diseases, is NLRP3. NLRP3, a cytosolic receptor, is one of 22 described human NLR family members; it functions to monitor the cytosol for stressful situations and has a crucial role in regulating immune responses (Kepp et al., 2011; Schroder and Tschopp, 2010; Shimada et al., 2012; Tschopp, 2011).

Once activated, the NLRP3 receptor induces NLRP3 oligomerization and recruitment of the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1, generating a multiprotein platform called the NLRP3 inflammasome. Then, NLRP3 activates caspase-1 and in turn drives the maturation of pro-inflammatory cytokines, such as IL-1β and IL-18. The NLRP3 inflammasome also conducts caspase-1-mediated cleavage of other substrates such as cytoskeletal proteins, glycolytic enzymes and caspase-7 (Tschopp, 2011; Zitvogel et al., 2012).

Mitochondria through ROS generation act as integrators across different stimuli activating the NLRP3 inflammasome (Kepp et al., 2011).

Increased levels of ROS are essential for NLRP3 activation; therefore, scavenging ROS with different chemicals suppresses inflammasome activation (Tschopp, 2011). Initially, Tschopp’s group, performing experiments in the human acute monocyte leukemia cell line, THP-1, suggested that NLRP3 inflammasome-activating ROS were generated by an NADPH oxidase stimulated upon particle phagocytosis, since in their model, ROS production was inhibited by NADPH oxidase inhibitors (Dostert et al., 2008).

However, later experiments performed by the same group (Zhou et al., 2011) and others showed that this result was ambiguous since immune cells from chronic granulomatous disease (CGD) patients, which have defective NADPH activity and thereby lack NADPH-dependent ROS generation, achieved normal levels of active caspase-1 and, in turn, active IL-1β.

Thus, these studies provided evidence of the existence of an alternative source of ROS (Meissner et al., 2010; van Bruggen et al., 2010; van de Veerdonk et al., 2010).

Since mitochondria are the main source of ROS, it is not surprising that mitochondria could also be the principal initiator of NLRP3 inflammasome activation. For example, the subcellular localization of NLRP3 suggests an important link between mitochondria and NLRP3. Under unstimulated conditions, most NLRP3 and ASC proteins are associated with the endoplasmic reticulum (ER).

Once the inflammasome is activated, NLRP3 redistributes to the perinuclear space and colocalizes with mitochondria (Zhou et al., 2011). Additionally, inhibition of voltage-dependent anion channels (VDACs), which are regulated by Bcl-2 family members through a decrease in mitochondrial Ca2 + levels and the resulting ROS production, significantly decreases NLRP3 inflammasome activation (Zhou et al., 2011).

Intriguingly, VDAC is also required for pro-apoptotic activity by Bax.
Other danger signals, specifically mtDNA and ATP, have been implicated in NLRP3 inflammasome activation; these signals can be released following different types of cell death (discussed below).

Indeed, it has been reported that mtDNA in the plasma of patients with femur trauma reaches values several thousand times higher than that of healthy volunteers, with functionally important immune repercussions (Zhang et al., 2010).

Additionally, mtDNA has been detected in RA synovial fluid and induces in vivo and in vitro inflammatory responses (Collins et al., 2004). Also, recent results have identified degraded mtDNA as a new DAMP subtype and a possible trigger of neurodegeneration (Mathew et al., 2012).

With regard to ATP, studies have shown that shedding of ATP from necrotic hepatocytes activates the NLRP3 inflammasome to generate an inflammatory microenvironment, alerting circulating neutrophils to adhere within liver sinusoids (McDonald et al., 2010).

Also, it has been shown that the release of extracellular ATP during apoptosis could be required for the successful immunogenic tumor cell death elicited by cancer treatment (Hahn et al., 2012).

Moreover, ATP can decrease intracellular K+ concentrations, a necessary event for NLRP3inflammasome activation, and mitochondria could also have a crucial role in modulating this process since they possess several K+ channels and can modulate intracellular K+ levels (Heinen et al., 2007).

Finally, NFPs is also considered a crucial mito-DAMP in several pathologies, including SIRS; this molecule can attract and stimulate neutrophils through high-affinity formyl peptide receptors (Zhang et al., 2010).

Interestingly, DAMPs and mito-DAMPs can activate antigenpresenting cells (APCs), as well as other nonimmune cell types, such as mesenchymal stem cells and astrocytes (Mathew et al., 2012; Pistoia and Raffaghello, 2011). In one study, degraded mtDNA strongly induced pro-inflammatory IL-1β, IL-6, MCP-1, and TNFα in mouse primary astrocytes, demonstrating the role of degraded mtDNA in inflammasome activation (Mathew et al., 2012).

The NLRP3 inflammasome and NF-κB pathways could work together to activate inflammatory cytokines, leading to overstimulation of the inflammatory response (Escames et al., 2011).

Once constituted, inflammasomes activate inflammatory caspases, mainly caspase-1, and subsequently induce IL-1β and IL-18 secretion. Then, pro-inflammatory cytokines can also activate NF-κB, generating a vicious cycle that increases the level and duration of the inflammatory response. The activation of inflammasomes may represent a possible explanation for this synergistic effect between mitochondrial impairment and cytokines.

Thus, studies investigating the role of mitochondria in inflammasome activation are revealing novel concepts in the development of inflammatory response, and these mechanisms may be of help in explaining the usual association of mitochondria with several inflammatory disorders and the aging process.

2.2.1. Role of mitochondria in the inflammatory response modulated by cell death.
Depending on the type of cell death, the dying cells could release and present at their surface specific signals that could modulate the type of immune response (Zitvogel et al., 2010).

Mitochondria have a key role in cell death (Kepp et al., 2011). Cell death can result from a variety of processes, including the apoptotic pathway and the necrotic pathway, 2 well-described extremes.

Apoptosis involves a series of orchestrated events requiring protein synthesis and ATP consumption. The dying cell forms vesicles that fragment into several apoptotic bodies; these are phagocytosed by macrophages or adjacent cells, thus avoiding the inflammatory reaction. Interestingly, contradictory recent data show that this paradigm is not always true, and depending on the inflammatory scenery, apoptosis could elicit a powerful inflammatory response (Shimada et al., 2012).

In particular, this study provides a link between apoptosis and inflammasome activation via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome. In this sense, Nakahira et al. also find that cyclosporine A, which modulates intrinsic apoptosis through inhibition of mitochondrial permeability transition, prevents the release of IL-1β by apoptotic stimuli (Nakahira et al., 2011).

Moreover, in vitro studies showed that silencing of antiapoptotic protein Bcl-2 increases IL-1β secretion while Bcl-2 overexpression resulted in the contrary (Shimada et al., 2012). In contrast, necrosis results from a rapid loss of cell homeostasis as intracellular ATP stores become depleted.

Cells increase their volume, leading to loss of cell membrane integrity and leakage of cellular components, thus inducing inflammation. In this study by Shimada above, necrotic stimuli did not secrete IL-1β (Shimada et al., 2012).

Mitochondrial DAMPs can also trigger autophagy. Autophagy is usually considered a type of cell death, although this process is thought to be an attempt to adapt and survive during periods of stress, such as nutrient deprivation, hypoxia, etc. (Edinger and Thompson, 2004).

Autophagy can control inflammation through macrophage-mediated clearing of apoptotic corpses or by inhibiting NLRP3 inflammasome activation by removing permeabilized or ROS-producing mitochondria (mitophagy). Thus, the inhibition of mitophagy results in spontaneous inflammasome activation (Goldman et al., 2010; Levine and Kroemer, 2008; Nakahira et al., 2011). Besides, mitochondria also play an important role in autophagia as they supply membranes for the biogenesis of autophagosomes (Hailey. 2010). Decreases in the expression of several autophagy-related genes, as well as the presence of mutations in these genes, have been found both in different inflammatory diseases, including neurodegenerative diseases, cardiovascular diseases, and Crohn’s disease (Gegg and Schapira, 2011; Travassos et al., 2010), and in the aging process (Salminen et al., 2012). For example, alterations in PINK1 and Parkin genes, which cooperate to identify and label damaged mitochondria for selective degradation via autophagy or mutations in ATG16 and IRGM, respectively, have been described. Additionally, the in vivo relevance of autophagic regulation of caspase-1 mediated inflammatory responses has been shown in two animal models of sepsis (the endotoxic shock model and the cecal ligation and puncture model of polymicrobial sepsis) (Nakahira et al., 2011). Furthermore, autophagy also declines during aging, generating the inflammatory condition via activation of inflammasomes, which in turn can actually accelerate the aging process (Salminen et al., 2012).

Mitochondria are triggers for the apoptotic pathway; several soluble pro-apoptotic proteins, including cytochrome c, are released from the mitochondrial intermembranous space after mitochondrial outer membrane permeabilization (MOMP). This leads to the formation of the apoptosome and the subsequent activation of the caspase cascade, which will trigger the execution phase of apoptosis. On the other hand, the mitochondrial permeability transition (MPT), which depends on the mitochondrial matrix protein cyclophilin D (Nakagawa et al., 2005), results in the instantaneous dissipation of the mitochondrial transmembrane potential and cessation of OXPHOS, thus rapidly increasing ROS, depleting ATP levels, and triggering necrotic cell death (Green et al., 2011; Hahn et al., 2012; Sasi et al., 2009).

As mentioned before, mtDNA can directly bind NLRP3; however, the current results do not rule out that mtDNA may bind to other members of the NLRP3 complex. ATP released from the dying cell constitutes a “find me” signal that acts as a chemo-attractant for monocytes and macrophages. Apoptosis releases much higher levels of cellular ATP related to necrosis (Hahn et al., 2012). It is therefore possible that ATP binding would be necessary for mtDNA binding (Shimada et al., 2012). DAMPs are often modified by processes of proteolysis and oxidation, which are related to cell death mechanisms. In this sense, HMGB1, a member of the high mobility group box family of DNA, can be released by necrotic cells, triggering a powerful immune response. During apoptosis, the generation of ROS by mitochondrial oxidized HMGB1 in turn enabled its immunostimulatory capacity (Hahn et al., 2012; Krysko et al., 2011).

Overall, depending on the specific circumstances and environment, cells can suffer from different lethal stress pathways promoting specific inflammatory responses, i.e., sterile inflammation or pathogen-induced inflammation. A better understanding of these processes is necessary for the development of new successful therapies to mitigate inflammatory diseases.

3. Pro-inflammatory mediators impair mitochondrial activity
A great number of inflammatory mediators, including the cytokines TNFα and IL-1β and the reactive nitrogen intermediate NO, may induce mitochondrial damage (López-Armada et al., 2006a; Maneiro et al., 2005; Stadler et al., 1992; Zell et al., 1997). TNFα and IL-1β decrease the activity of MRC complex I, ATP production, and mitochondrial membrane potential (Δψm). These mediators also induce the accumulation of significant amounts of ROS (Guidarelli et al., 2007; Kim et al., 2010). Interestingly, complex I together with complex III have been suggested to be major sources of ROS (Guzy et al., 2005), and the activities of complexes II and IV were also decreased in other cell types (Biniecka et al., 2011; Maneiro et al., 2003). Moreover, in adipocytes, the changes induced by TNFα cause pronounced morphological changes in the mitochondria, presumably due to variations in the levels of several mitofusion proteins (Chen et al., 2010). Another important study by Rowlands et al. showed that the severity of inflammation in mouselung microvessels is modulated by the inhibitory effect of TNFR1 ectodomain shedding by mitochondrial Ca2+ (Rowlands et al., 2011). Thus, mitochondrial Ca2+ and ROS are primary mediators of TNFαmediated inflammatory responses (Dada and Sznajder, 2011).

NO is another particularly important mediator in the pathophysiology of inflammatory processes. A variety of NO donors have been shown to suppress mitochondrial energy production in different cell types. Moreover, most of the catabolic effects of NO are potentially related to the ability of NO to combine with the superoxide anion to generate peroxynitrite, which, as mentioned earlier, is a powerful oxidant capable of inhibiting important enzymes and affecting mitochondrial integrity (Fermor et al., 2010; Johnson et al., 2000; Maneiro et al., 2005). In particular, NO induced a significant, reversible decrease in the activity of complex IV as well as a reduction in Δψm (Tomita et al., 2001). NO can also irreversibly inhibit respiration, most likely through ATP synthase and the strong oxidant peroxynitrite, which inactivates all respiratory complexes. Other studies demonstrated that, in intact U937 cells, peroxynitrite enhances the formation of superoxide ions in the mitochondria through a Ca2+-dependent mechanism that involves the inhibition of complex III, which then dismutates to H2O2 (Guidarelli et al., 2007).

Mitochondria play an important role during apoptosis. As mentioned above, severe mitochondrial damage can even lead to cell death by necrosis or by activation of the apoptotic signaling pathway and related caspases (Cillero-Pastor et al., 2011; Irrinki et al., 2011; López-Armada et al., 2006b; Maneiro et al., 2005). Indeed, the classic signs of cell death are preceded by mitochondrial alterations, which include changes in MRC activities, loss of Δψm, decreases in energy production, and/or induction of the mitochondrial permeability transition (MPT), and all of these alterations are modulated by both cytokines and NO. Thus, such pro-inflammatory mediators can modulate cell survival by exerting their effects on mitochondria. In addition, mito-DAMPs can be generated as consequence of these cell death processes.

To summarize, mitochondrial complexes could suffer permanent alterations caused by mutations in DNA or inflammatory mediators, such as reactive nitrogen species (RNS). Other stimuli, including cytokines and NO, induce functional and reversible modification of mitochondrial complexes at physiological concentrations. The combination of both effects (permanent and functional alteration of mitochondrial complexes) may cause a more substantial modification of mitochondrial activity, establishing continual, sublethal mitochondrial damage and thereby generating a vicious cycle of increasing inflammation that is difficult to break. Therefore, preserving mitochondrial function could reduce excessive oxidative stress and may represent a novel therapeutic advantage for patients with inflammatory diseases and chronic low-grade inflammation in aging.

4. Physiological function of ROS in the regulation of cell signaling
Although ROS has been traditionally linked to disease states, its crucial roles in normal physiological processes (vascular tone, oxygen sensing, and skeletal muscle physiology) and protective mechanisms, such as the above-mentioned host defense system and control of inflammation and immune responses, are becoming obvious. In this sense, there is a tight balance between appropriate redox states and oxidative stress. The final balance results from the magnitude and class of ROS, as well as the persistence of ROS production. Thus, high ROS concentrations may drive unspecific damage to nucleic acids, proteins, carbohydrates, and lipids or overtake the signaling pathways regulated by ROS.

In contrast, low to moderate levels of ROS lead to physiological regulation of cell signaling pathways (protein phosphorylation, ion channels, and transcription factors) and subsequently control cellular processes, such as differentiation, apoptosis, and migration.

A brief description of the most relevant physiological roles of ROS is as follows. ROS participate in immune system, at both innate and acquired levels. These molecules produced by phagocytes represent one of the early actions of defense against pathogen invasion. Notably, some pathogens have been shown to downregulate mitochondrial ROS generation and thereby prevent macrophage defense mechanisms, favoring pathogen persistence (Basu Ball et al., 2011). An example of the importance of ROS in immune system signaling is reflected in chronic granulomatous disease (CGD), which is caused by a lack of ROS-generating phagocytes. In this manner, CGD patients inefficiently kill invader bacteria by ROS, suffering recurrent infections (Mauch et al., 2007). Interestingly, CGD patients and mice defective for ROS production also show evidence of noninfectious inflammatory states, supporting the role of ROS in controlling inflammation and immune responses (van de Veerdonk et al., 2010). With respect to acquired immunity, ROS participates in signal transduction cascades within T lymphocytes (Alfadda and Sallam, 2012). Furthermore, as detailed earlier, recent evidence highlights a role for ROS in immune responses through NRPL3 activation (Nakahira et al., 2011; Shimada et al., 2012; Zhou et al., 2011).

Other illustrations of the crucial role of ROS in normal physiological processes are the key regulation of vascular system, the role of ROS in oxygen sensing through stabilization of hypoxia-inducible factor-1 (HIF-1) and the subsequent stimulation of angiogenesis and bioenergetic responses to rescue redox homeostasis, the role of ROS in skeletal muscle physiology regulation through glucose uptake during contraction, and the role of ROS in regulating gene stability, transcription, and signal transduction (Alfadda and Sallam, 2012).
Finally, increasing evidence that antioxidants may be harmful highlights the physiological role of ROS. Specifically, Ristow et al. reported that antioxidant supplementation may prevent increased insulin sensitivity by reducing the expression of ROS-sensitive transcriptional regulators of insulin sensitivity associated with exercise training in humans (Ristow et al., 2009). Also, the nuclear factor-erythroid 2-related factor 2 (Nrf2), a redox stress-sensitive transcription factor that induces several antioxidant and detoxification genes, is kept inactive in the absence of redox stress by its binding to Kelch-like ECH-protein 1 (Keap1). Thus, disruption of Nrf2 signaling impaired angiogenesis and microvascular rarefaction in aging (Valcarcel-Ares et al., 2012). In this sense, it is worth bearing in mind that antioxidant strategies will likely not be successful because their activity is too unspecific, too insufficient, and too delayed; however, it is also plausible that the ineffectiveness of antioxidant strategies may arise from their inhibition of essential cell functions that require ROS.

However, excessive levels of ROS cause not only oxidative damage to macromolecules, but can also affect the physiological signaling pathways regulated by ROS. Among other consequences, increased ROS production will lead to augmented activation of the transcription factors HIF-1, NF-κB, and activator protein-1 (AP-1) as well as the NLRP3 inflammasome. This will increase the release of pro-inflammatory cytokines that in turn will enhance ROS production, and hence, feed-back in a self-stimulatory manner, thereby amplifying the inflammatory response (Dröge, 2002).

5. Mitochondrial dysfunction and inflammatory responses in chronic human pathologies
Extensive research over the past decade has revealed that continued mitochondrial dysfunction can lead to chronic inflammation, which in turn may mediate most chronic diseases, including rheumatoid, cardiovascular, neurological, or metabolic diseases.

5.1. Rheumatoid diseases
Osteoarthritis (OA) and RA are the most common rheumatoid pathologies. In both of these conditions, the mitochondrial characteristics, including MRC activity, ATP synthesis, and Δψm, are adversely affected in several cell types. In particular, ex vivo studies have revealed that OA chondrocytes, as compared with normal chondrocytes, showed significant decreases in the activities of MRC complexes II and III and Δψm (Johnson et al., 2000; Liu et al., 2010; Maneiro et al., 2003). In RA and

systemic juvenile idiopathic arthritis, studies have demonstrated that there is a deficiency in a subunit of the MRC complex IV in the synovium (Biniecka et al., 2011; Ishikawa et al., 2009). Furthermore, human RA synoviocytes (Da Sylva et al., 2005; Ospelt and Gay, 2005) and normal synoviocytes under arthritis-like conditions, such as hypoxia (Biniecka et al., 2011), which causes a significant increase in Δψm in synovial cells, have been shown to exhibit significantly higher mutation rates than normal-aged or OA synoviocytes. These data support the conclusion that mitochondrial mutagenesis is correlated with the local inflammatory environment in arthritis (Harty et al., 2011). Other cells, such as T lymphocytes, show mitochondrial impairment in patients with RA and systemic lupus erythematosus (Gergely et al., 2002; Moodley et al., 2008).

Oxidative stress can contribute to the activated phenotype of synoviocytes in inflammatory arthritis and OA chondrocytes (Davies et al., 2008; Filippin et al., 2008; Henrotin et al., 2005). Interestingly, studies have shown that inhibition of complex III or V induces ROS production and NF-κB activation in human articular chondrocytes (CilleroPastor et al., 2008; Milner et al., 2007) and synovial cells (Valcárcel-Ares et al., 2010) in vitro. Additionally, inhibition of complex III or V in chondrocytes/synoviocytes induces low-grade inflammatory and matrix degradation processes through the synthesis of pro-inflammatory stimuli, including prostaglandin E2 (PGE2), the chemokines IL-8 and monocyte chemotactic protein-1, and several matrix metalloproteinases (MMPs) (Cillero-Pastor et al., 2010; Cillero-Pastor et al., 2008; VaamondeGarcía et al., 2012).

Specifically, it has been shown that the inhibition of MRC activity induces cyclooxygenase-2 (COX-2) expression and PGE2 production through redistribution of mitochondrial Ca2+, generation of ROS, and activation of the transcription factor NF-κB (Biniecka et al., 2010; Cillero-Pastor et al., 2008). Mitochondrial dysfunction can also induce PGE2 liberation in human OA chondrocytes through 4-hydroxy-2nonenal (4-HNE) (Vaillancourt et al., 2007), a lipid peroxidation byproduct that is associated with a higher frequency of mtDNA mutations, which are often increased in articular tissues of patients with inflammatory arthritis (Biniecka et al., 2010; Vaillancourt et al., 2007).

In addition to the above processes, the decline in mitochondrial function can also increase the inflammatory responsiveness of both normal human chondrocytes and synoviocytes to cytokines, promoting mechanisms that may contribute to joint destruction and pain (Valcárcel-Ares et al., 2010; Vaamonde-García et al., 2012). Interestingly, the hypoxic state of arthritic joints in vivo, which correlates with increased disease activity and mtDNA mutagenesis rates, exacerbates the inflammatory response in synoviocytes by increasing COX-2 expression and the release of MMPs in response to IL-1β (Demasi et al., 2004). In fact, inflammasome activation may explain these synergistic phenomena. NF-κB signaling and the NLRP3 inflammasome pathway could work together to activate inflammatory cytokines, thereby leading to overstimulation of the inflammatory response (Escames et al., 2011). As additional support for the critical role of the inflammasome in the pathogenesis of OA, the NLRP3 inflammasome could also mediate the pathological effects of hydroxyapatite crystals in vitro and in vivo (Jin et al., 2011).

Other DAMPs/mito-DAMPs are also highly secreted in tissues of rheumatoid patients. In fact, oxidized mtDNA, which has immunostimulatory properties and is capable of inducing arthritis in mice, can be detected in the synovial fluid of RA patients, but not in that of control subjects (Collins et al., 2004). Some studies have also considered cytochrome c a mitoDAMP that can induce arthritis (Pullerits et al., 2005); however, recent data suggest that this implication is not mediated by a direct role in NLRP3 activation (Shimada et al., 2012). Moreover, as mentioned earlier, autophagy prevents the accumulation of dysfunctional mitochondria. In this regard, OA cartilage has been shown to be deficient in autophagic processes, and the pharmacological activation of autophagy may be an effective therapeutic approach for OA (Caramés et al., 2012). Together, these findings support the critical role of mitochondrial dysfunction in rheumatoid disorders.

5.2. Cardiovascular diseases
Abnormalities in mitochondrial functions are increasingly recognized in association with cardiomyopathies. In fact, the myocardium is a highly energy-demanding tissue, with mitochondria supplying greater than 90% of ATP. Consequently, mitochondria are essential for cardiomyocyte function and viability (Dutta et al., 2012). Since ROS are constantly generated during mitochondrial respiration, it is not surprising that a dysregulation in the production of free radicals can cause oxidative damage in the heart. This oxidative damage may cause mtDNA mutations that result in mitochondrial dysfunction. Moreover, increased mitochondrial Ca2 + levels modulate myocardial inflammation and dysfunction in states of injury, such as sepsis and burn trauma (Maass et al., 2005). Another important study showed that escape of mtDNA from autophagy-mediated degradation leads to TLR9-mediated inflammatory responses in cardiomyocytes and may induce myocarditis and dilated cardiomyopathy (Oka et al., 2012).

With advanced age, mitochondrial ROS production significantly increases both in the heart (Judge et al., 2005) and vasculature (Ungvari et al., 2007), making the vascular endothelium and smooth muscle susceptible to ROS oxidation. In fact, mitochondria-derived ROS likely contribute to the development of chronic low-grade vascular inflammation in aging by activating redox signaling pathways (Dai et al., 2012). This low-grade vascular inflammation actives the redox-sensitive transcription factor NF-kB, stimulating endothelial activation by production and secretion of cytokines, growth factors, and proteases in the vascular wall, thereby promoting atherosclerosis. This damage is augmented by excessive ROS generation in the heart and the vasculature, as well as defective oxidant scavenging. These mechanisms disturb endothelial homeostasis (i.e., induce endothelial dysfunction), a characteristic feature of patients with coronary atherosclerosis (Munzel et al., 2010).

Traditionally, it has been hypothesized that cardiovascular diseases are precipitated by atherosclerosis due to arterial blockage from fatty deposits. However, in recent years, diagnostic and therapeutic strategies have been based not only on control of cholesterol levels, healthy diet, and smoking, but also taking into account other factors, such as inflammatory markers and mitochondria (Garg, 2011; Marchant et al., 2012). In particular, the roles of inflammasomes in the development of cardiovascular diseases are currently being studied in depth (Garg, 2011). Finally, pathways that improve mitochondrial function and attenuate mitochondrial oxidative stress (i.e., antioxidant therapies) show efficacy in various animal models and promising results for the treatment of cardiovascular diseases.

5.3. Neurological diseases
Increasing evidence has demonstrated the role of mitochondrial dysfunction in neurological and neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), dementia, multiple sclerosis, ataxia, and encephalomyopathies (Monje et al., 2003; Morán et al., 2012; Witte et al., 2010). A common feature in neurodegenerative diseases is the presence of neuroinflammation and oxidative stress. In both AD and PD, oxidative stress activates inflammatory signaling pathways through several mechanisms, including exacerbating the production of ROS, inducing mitochondrial dysfunction, activating microglia and astrocytes to release pro-inflammatory cytokines, and others (Schapira, 2010; von Bernhardi and Eugenín, 2012; Whitton, 2007). In fact, animal models of PD, induced by the mitochondrial inhibitor rotenone, exhibit increased production of IL-1β in the hypothalamus (Yi et al., 2007).

Furthermore, pro-inflammatory cytokines released in activated glia have been shown to stimulate increased ROS production in the brain, contributing to other mechanisms that amplify ROS production and lead to deterioration of mitochondrial function. In addition to exacerbating disease progression, this process may also suppress neurogenesis. Severe mitochondrial damage may even lead to cell death by necrosis or by activation of apoptotic signaling and activation of caspases (Voloboueva and Giffard, 2011).

Because neurodegenerative diseases are accompanied by chronic inflammation, the induction of mitophagy may have beneficial effects by removing permeabilized or ROS-producing mitochondria (Green et al., 2011). Moreover, as mentioned earlier, alterations in several genes involved in autophagy, such as PINK1 or Parkin, have been described in PD. Furthermore, downregulation of mitochondrial ROS may also decrease astrocyte activation (Voloboueva and Giffard, 2011). Finally, it has been recently described in mouse primary astrocytes how degraded mtDNA could be a new identified subtype of DAMP inducing inflammasome activation and ensuing neurodegeneration (Mathew et al., 2012).

5.4. Metabolic diseases
Insulin resistance, pancreatic islet β-cell failure, and elevated plasma free fatty acid (FFA) levels are features commonly associated with the development of several metabolic diseases, including obesity, type 2 diabetes mellitus, and metabolic syndrome. Accumulating evidence indicates that mitochondrial dysfunction is a central contributor or mediator of these processes (Coletta and Mandarino, 2011; Ma et al., 2012; Martins et al., 2012; Vial et al., 2010).

Diverse sources of inflammation can contribute to the insulin resistance observed in obesity and type 2 diabetes. Elevated plasma FFA levels play an important role in the development of skeletal muscle insulin resistance through different mechanisms, including oxidative stress, inflammation, and mitochondrial dysfunction (Coletta and Mandarino, 2011; Martins et al., 2012). The inflammation induced by FFAs may induce impairment in mitochondrial function in adipocytes (Ji et al., 2011; Youssef-Elabd et al., 2012). Furthermore, FFAs can activate these inflammatory signaling pathways directly through interactions with members of the TLR family and inflammasome activation (Reynolds et al., 2012; Wen et al., 2011; Youssef-Elabd et al., 2012).
Since mitochondrial dysfunction drives NLRP3 inflammasome activation, it is not surprising that this pathway can underlie cellular metabolic disorders. Specifically, a large body of evidence demonstrates a pathological role for the association of NLRP3 with a nonmitochondrial protein, thioredoxin-interacting protein (TXNIP), in type 2 diabetes and obesity. For example, TXNIP deficiency impairs activation of the NLRP3 inflammasome, improving glucose tolerance and insulin sensitivity in β-cells and adipocytes and decreasing fat deposits in the liver (Vandanmagsar et al., 2011; Zhou et al., 2010). Interestingly, high levels of oxidized DNA have been related with diabetes (Simone et al., 2008). It is possible that the elevated levels of damaged DNA detected in diabetes could be related with the activation of NLRP3 (Shimada et al., 2012). Consequently, mitochondria may represent novel pharmacological targets for therapeutic interventions in metabolic disorders.

5.5. Cancer and immunogenic cell death
Mitochondrial processes play an important role in tumor initiation and progression. Otto Warburg made the first association between mitochondrial dysfunction and cancer in the 1930s. He observed increased rates of aerobic glycolysis in a variety of tumor cell types, and he hypothesized that the cell’s choice of the glycolytic route to produce ATP instead of the more productive oxidative phosphorylation route may be due to the impaired respiratory capacity of these cells (Warburg, 1956). Years after this discovery, a number of cancer-related mitochondrial defects have been identified. In this sense, altered expression and activity of respiratory chain subunits as well as mtDNA mutations have been associated with human tumors. For example, MRC Complex I dysfunction has been associated with the pathogenesis of Hürtle cell tumors of the thyroid (Máximo et al., 2005). In the same way, low activity of Complex III has been found in breast cancer (Putignani et al., 2008) and Hürtle cell tumors (Bonora et al., 2006; Stankov et al.,

The aggressiveness of renal cell tumors is related to lower Complex II, III, and IV activities (Simonnet et al., 2002). As we know, dysfunction in MRC complexes is associated with mtDNA mutations. In fact, mutations in mtDNA have been reported in a variety of cancers, including ovarian, thyroid, salivary, kidney, liver, lung, colon, gastric, brain, bladder, head and neck, and breast cancers, as well as leukaemia (Modica-Napolitano and Singh, 2004).

In the context of mitochondrial dysfunction, we found that ROS is a key player in tumor formation and progression. It is now recognized that ROS plays an important role as a signaling molecule, mediating changes in cell proliferation, differentiation, migration, and invasiveness, as well as large-scale changes in gene transcription (Weinberg and Chandel, 2009). It is well known that mitochondrial dysfunction increases ROS levels, and tumor cells generally exhibit higher levels of ROS than normal cells (Weinberg and Chandel, 2009). One signaling pathway that may be particularly important to ROS-mediated tumorigenesis involves the activation and stabilization of HIF. HIF is a key transcription factor in cancer progression that regulates the enhancement of both glucose metabolism and angiogenesis. HIF activation occurs under hypoxic conditions, but ROS also appears to stabilize HIF under conditions of normal oxygen concentrations, leading to aberrant activation of HIF and promoting tumorigenesis (Chandel et al., 1998).

In addition, it has been demonstrated that tumors lacking the tumor-suppressor succinate dehydrogenase (Complex I) and fumarate hydratase exhibit impaired HIF degradation (King et al., 2006), once again linking mitochondrial impairment with oncongenesis.
As was discussed earlier, mitochondrial ROS production results in activation of the NLRP3 inflammasome with the consequent release of pro-inflammatory cytokines (Zitvogel et al., 2012). In vivo experiments suggest that inflammasome products, such as IL-1β, can directly drive oncogenesis or suppress immunosurvellaince mechanisms, thereby facilitating tumor development (Zitvogel et al., 2012). In the same way, many human cancers are etiologically linked to chronic inflammatory processes, such as those that occur with gastric cancer, hepatic cancer, and colorectal cancer (Coussens and Werb, 2002). However, the role of the inflammasome in carcinogenesis is controversial. Yet, in sharp contrast, the inflammasome or its products, depending on the specific tumor and its grade, can reduce tumorogenesis by causing cell death and promoting antitumor immune responses (Zitvogel et al., 2012).

In this context, recently developed therapies have been able to induce cell death by triggering ROS production. These include a specific group of mitochondrial-targeted anticancer drugs (mitocans) (Hahn et al., 2012). An example of these mitocans is the vitamin E succinate. This pro-oxidant selectively targets cancer cell mitochondria and is a potent inducer of ROS, leading to apoptosis in cancer cells but not in related normal cells. This is because cancer cells are under much more oxidative stress than normal cells and thus are more vulnerable to further damage by ROS-generating agents. Interestingly, these types of therapies have immune-enhancing properties, meaning that they are able to modulate antitumor immune responses, stimulating the immune system to cause immunogenic tumor cell death by upregulating the release of DAMP molecules. Of note, the activation or inhibition of the inflammasome as cancer therapy will likely depend on the specific cancer and its grade (Hahn et al., 2012).

6. Mitochondrial dysfunction and inflammatory responses in acute human pathologies
Generally, the acute inflammatory response to infection and tissue damage should be able to reduce injury to the organism, preventing further damage to the cells or tissues. However, as mentioned above, when the acute inflammatory responses cannot be effectively resolved, progressive damage and multi-organ failure can occur. Sepsis, a representative acute inflammatory disease, is unfortunately the main cause of death in critical care units. Mitochondrial dysfunction and impaired oxygen consumption play a role in sepsis, and the severity of sepsis has

been shown to correlate with mitochondrial damage in both humans and experimental models (Garrabou et al., 2012; Kung et al., 2012). Thus, changes to the ultrastructure of the mitochondria and significant inhibition of mitochondrial complex activities have been described in several cell types isolated from septic patients. Also, during overwhelming sepsis, increased Ca2+ influx into the mitochondria leads to mitochondrial dysfunction, resulting in the release of cytochrome c and cell death (Dada and Sznajder, 2011).

In this context, as mentioned before, mitochondrial damage may release several mito-DAMPs, including mtDNA. Consistent with this, septic plasma samples have shown significantly increased amounts of mtDNA and inflammatory cytokines, with corresponding NF-κB activation, and a correlation between adverse outcomes in sepsis and levels of extracellular mtDNA has been demonstrated (Garrabou et al., 2012). Since mtDNA can activate the NLRP3 inflammasome, its participation in the pathogenesis of sepsis is not surprising.

Interestingly, NLRP3 polymorphisms may be used as relevant risk estimates for the development of sepsis, which highlights the importance of NLRP3 in the pathogenesis of sepsis (Zhang et al., 2011). As expected, mitochondrial antioxidants have been shown to alleviate oxidative and nitrosative stress in several in vitro and in vivo model of sepsis (Apostolova et al., 2011; Zang et al., 2012). Taken together, these findings have led to a better understanding of the pathophysiological
processes of acute diseases and will be fundamental in improving the outcome of sepsis-related disorders.

7. Mitochondrial protection attenuates inflammation
Excessive mitochondrial oxidative stress plays a central role in triggering the deleterious cascade of events associated with inflammatory diseases and chronic low-grade inflammation in aging. In this regard, several approaches to preserve mitochondria are under investigation, including the use of antioxidant compounds, development of specific mitophagy-inducing therapies, and the pharmacological manipulation of mitochondrial sirtuins (SIRTs).

Strategies that control ROS production could limit either the redox-sensitive inflammatory pathway or the direct activation of the inflammasome. Thus, several meta-analyses have supported that dietary antioxidant intake is associated with a lower incidence of inflammatory diseases (Alissa and Ferns, 2012; Demetriou et al., 2012; Lahiri et al., 2012; Patelarou et al., 2011). In particular, resveratrol, a natural antioxidant found in grape skin and red wine, improves mitochondrial function by preventing oxidative stress and subsequent inflammation (Catalgol et al., 2012). In vitro, this molecule attenuates mitochondrial oxidative stress in several cell types, i.e., by restoring mitochondrial

complex III activity, which is considered to be the major source of mitochondrial oxidative stress (Xu et al., 2012). Moreover, resveratrol pretreatment efficiently prevents inhibition of mitochondrial membrane depolarization and ATP depletion, preserving mtDNA content and inhibiting COX-2 activity through a decrease in NF-κB activation in different cells (Dave et al., 2008; Lin et al., 2011; Ungvari et al., 2009; Xu et al., 2012). In animal models, resveratrol protects from developing age-related and acute diseases by improving mitochondrial function (Elmali et al., 2007; Jian et al., 2012; Lin et al., 2012; Ungvari et al., 2010).
As described earlier, an imbalance between mitophagy and mitochondrial biogenesis could be involved in inflammatory pathology. Autophagy/mitophagy decreases ROS production and mtDNA release after MPT opening, in turn affecting the NF-κB pathway and inflammasome activation. Thus, pharmacological induction of autophagy could mitigate inflammatory reactions. For example, activation of autophagy by rapamycin reduces the severity of experimental OA (Caramés et al., 2012). Interestingly, resveratrol also improves mitochondrial function through induction of mitochondrial biogenesis (Csiszar et al., 2009; Ungvari et al., 2009).

Recent studies revealed that SIRTs or class III histone deacetylases, have an essential role in mitochondrial protection against oxidative stress (Pereira et al., 2012). Three of the 7 SIRTs described in humans, SIRT3, SIRT4, and SIRT5, are localized inside mitochondria, which lends support to the potential role of SIRTs in mitochondrial biology (Hirschey et al., 2010). SIRTs can indirectly regulate the expression of several inflammatory genes through deacetylation of several signaling proteins, including the transcription factors FOXO (Salminen et al., 2008) and NF-κB (Natoli, 2009) and the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) (Sugden et al., 2010). Deacetylation of FOXO by SIRT1 or SIRT3 upregulates the expression of SOD2 and catalase, thereby decreasing cellular ROS levels. Also, deacetylation of PGC-1α by both SIRT1 and SIRT3 stimulates mitochondrial biogenesis (Pereira et al., 2012). As mentioned above, several studies have demonstrated that resveratrol can protect mitochondrial functions through activation of SIRTs (Shinmura et al., 2011).

While the role of mitochondria in the development of the inflammatory response is now beginning to be understood, there is still a lot to be discovered. Understanding the mechanisms that induce inflammation through mitochondrial impairment may help to identify relevant therapeutic targets for the treatment of multiple degenerative and acute disorders as well as the aging process. For these reasons, it is plausible that, in few years, specific mitochondrial-targeting pharmacological treatments of inflammatory responses may become a reality. Indeed, some pharmaceutical companies have already begun designing new molecules that are the equivalent of super-antioxidants and have demonstrated exciting results in several preclinical studies in vitro and in vivo (McManus et al., 2011; Mukhopadhyay et al., 2012).

8. Limitations and future research directions
The use of effective strategies to increase our knowledge about the role of mitochondrial dysfunction in the inflammatory components of multiple degenerative and acute diseases, as well as in the aging process, is necessary for the development of new successful therapies aimed at improving human health. Thereby, an appropriate way to avoid contradictory results is the use of unmanipulated cells from healthy and diseased donors. Most in vitro studies are conducted in isolated cells kept in standard media with high glucose concentrations and under aerobic conditions, quite different from effects in tissues in a live organism, which could lead cells to obtain their energy predominantly from anaerobic glycolysis. Thus, the hypoxic nature of rheumatoid joints in vivo, which is correlated with increased mtDNA mutagenesis rate and disease activity, increases the inflammatory response in synoviocytes by increasing COX-2 expression and MMPs release in response to IL-1β (Demasi et al., 2004). For this reason, it is likely that the effects of mitochondrial impairment observed in cultures

maintained in normal atmosphere will even be more crucial in damaged tissue, where mitochondrial activity is probably lower due to genetic predisposition, somatic mutations in the mtDNA of the MRC as a consequence of the elevated levels of pro-inflammatory mediators (notably ROS), or the direct effects of pro-inflammatory mediators on the MRC. Future in vitro studies must take this into account. In addition, an important tool in these studies could be the transmitochondrial cybrid system, which allows the study of the effects of mitochondrial dysfunction and inflammatory responses with a common nuclear background, thereby avoiding the large number of inflammatory properties regulated by nuclear-encoded genes (Kaipparettu et al., 2010).

The majority of studies to date were conducted using in vitro or ex vivo strategies. Thus, it is necessary to translate the results to in vivo designs to support a clear role for mitochondrial dysfunction in the development of the inflammatory response. Animal models are also useful tools in medical science research. In this sense, different studies have reported that transgenic expression of mitochondrial-targeted catalase in mouse models of chronic diseases, such as cancer or Alzheimer’s disease, reduced primary tumor invasiveness and decreased metastasis incidence and burden (Goh et al., 2011) and several typical events of the disease (Mao et al., 2012).

Finally, in transgenic mouse lines, the overexpression of catalase targeted to mitochondria reduced age-associated pathologies and led to an extension in lifespan (Schriner et al., 2005). Overall, these in vivo studies support that mitochondria-targeted molecules may be an effective therapeutic approach to treat or prevent inflammation linked to numerous degenerative and acute diseases. In addition, other interesting models to be kept in mind are represented by mice with increased mtDNA mutations that exhibit pathology associated to human aging (Trifunovic, 2006) Analysis of the inflammasome has not been performed in these animals, but could be a useful model in which to study the role of mitochondria in NLRP3 inflammasome activation.

However, despite our increasing knowledge on the role of mitochondria in the development of the inflammatory response, there is still much to learn. Several key questions remain to be addressed. For example, which mechanisms determine whether mitochondria induce the inflammasome or apoptosome? Along these lines, recent research has suggested that oxidized mtDNA released during apoptosis results in activation of the NLRP3 inflammasome, providing a link between apoptosis and inflammasome activation (Shimada et al., 2012).

Interestingly, VDAC, whose activity is modulated by Bcl-2 family members, is decisive for inflammasome activation, and also in apoptosis induction by Bax (Yamagata et al., 2009; Zhou et al., 2011). However, what is the exact mechanism through which mtDNA activates the NLRP3 inflammasome? It also remains to be determined the importance of inflammasome in non-immune cell types.

Furthermore, other factors, such as tissue specificity, the effects of environmental factors, and age, may determine the final outcome. Clearly, more work is necessary to shed light on how mitochondrial dysfunction modulates the inflammatory response associated with distinct degenerative and acute diseases as well as the aging process.

9. Conclusions

Both acute and chronic inflammatory diseases, as well as the aging process, have been linked to accumulation of ROS and RNS, which could potentially be a main source of mitochondrial genomic instability leading to respiratory chain dysfunction. Indeed, mitochondrial impairment could modulate innate immunity through both redox-sensitive inflammatory pathways and direct activation of the inflammasome. Mitochondria could integrate these 2 pathways, leading to an overstimulation of the inflammatory response (Fig. 1). In conclusion, there is substantial evidence supporting the hypothesis that a decline in mitochondrial function is essential to the development of the inflammatory phenotype observed in multiple human degenerative or acute diseases as well as in advanced age.

All of these findings support mitochondria as new pharmacological targets. The preservation of mitochondrial function could reduce oxidative stress and may represent a novel therapeutic advantage for patients with degenerative or acute diseases as well as functional decline in aging. Despite these advances in our knowledge of inflammatory alterations, it is necessary to keep in mind that the best treatment is prevention; this is true for both inflammatory-related diseases and in healthy aging. Although we are becoming increasingly aware of the importance of a healthy diet, there is still a long way to go. Adequate nutrition could in fact be the best way to prevent aging declines and inflammatory degenerative or acute diseases. Thus, as Hippocrates said 25 centuries ago, “Let food be the medicine and medicine be the food”