Mitochondrial dysfunction acts as a modulator of the immunometabolic route for activating the cytosolic DNA sensor pathway in triggering innate immunosurveillance

Abstract

Mitochondria, in addition to their classic role in energy production, have emerged as central hubs in the regulation of innate immunity. Under conditions of cellular stress, mitochondrial dysfunction triggers the release of mitochondrial DNA (mtDNA) into the cytosol or extracellular space, activating potent inflammatory pathways such as cGAS-STING, NLRP3 and TLR9. mtDNA release, driven by factors such as oxidative damage, membrane permeabilization, and various cell death pathways, is involved in immune surveillance and the pathogenesis of various diseases. At the same time, this downstream event leads to profound reorganization of immune cell metabolism, influencing functional polarization and inflammatory outcomes. This review presents the mitochondrion as an interface between metabolism, immunity, immunometabolites, and danger signalling. We explore the molecular mechanisms of mtDNA release, its conversion into immune signals, and its impact on metabolism in immune cells. Translational implications for pathologies such as neurodegenerative, autoimmune, and neoplastic diseases are also discussed. Deciphering the interconnection between mitochondrial stress, mtDNA release, and immunometabolic rewiring could open new avenues for the treatment of complex diseases and drive innovation in immunotherapy and regenerative medicine.

Introduction

Mitochondria are not only central to cellular energy metabolism (oxidative phosphorylation (OXPHOS) and ATP production) but also serve as key regulators of innate immunity by modulating inflammatory signalling and acting as a major source of damage-associated molecular patterns (DAMPs), including mitochondrial DNA (mtDNA) and reactive oxygen species (ROS) [1].

Under cellular stress, mitochondrial integrity is compromised, leading to the release of mtDNA into the cytosol. Owing to its structural differences from nuclear DNA, mtDNA functions as a potent DAMP [2], establishing a molecular link between mitochondrial dysfunction, immunometabolic reprogramming, and innate immune activation. Understanding these interconnected processes is essential for elucidating how mitochondrial stress drives sterile inflammation and contributes to chronic disease pathogenesis [3].

The relationship between mitochondrial health and immune function is particularly evident in macrophages, key effectors of innate immunity. These cells exhibit remarkable plasticity, polarizing into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes in response to environmental cues [4]. This functional diversity is closely tied to metabolic programming. Mitochondrial dysfunction disrupts this balance, driving metabolic rewiring, accumulation of inflammatory intermediates such as succinate, and increased ROS production. Importantly, impaired mitochondrial integrity and mtDNA release amplify innate immune signalling and influence macrophage polarization.

Herein, we investigate the intricate interplay between mitochondrial dysfunction, DAMP signalling, and immunometabolic reprogramming that underlies the pathogenesis of numerous disorders. Evidence indicates that mitochondrial impairment in immune cells, particularly macrophages, exacerbates inflammation in diverse conditions, including cardiovascular injury [5], ischemia-reperfusion damage [6], infectious diseases [7], immunodeficiency diseases [3] and cancer [2, 8]. Deciphering how mitochondrial dysfunction governs macrophage metabolism and polarization is therefore critical for understanding innate immunosurveillance and developing novel therapeutic strategies for chronic inflammatory and immune-mediated diseases.

Role of metabolism in cell polarization of innate immune response

As the most important innate immune cells and effective antigen-presenting cells, macrophages are remarkably versatile. By identifying risk factors, they initiate the natural immune response; conversely, they modify host immunity by polarizing into different phenotypes in response to microenvironmental changes. Furthermore, the host’s immunological homeostasis depends on the delicate balance of macrophages in various polarization states, each of which performs a variety of activities [9]. In order to reduce inflammatory disorders, it is therefore of great importance to modify macrophage activation by encouraging the repolarization of M1 macrophages to M2 macrophages [10]. In order to assure effective microbial death, M1 macrophages generate reactive oxygen and nitrogen species, secrete pro-inflammatory cytokines, and exhibit increased expression of MHC-I/II, CD80, and CD86. On the other hand, persistent M1 activation might result in chronic inflammation and collateral tissue damage. Additionally, a variety of non-inflammatory stimuli can activate macrophages. In terms of function, M2 macrophages mediate Th2cytokines-driven diseases, encourage tissue repair, and reduce Th1/M1-driven inflammation. M2 macrophages are distinguished at the molecular level by a variety of distinct marker genes, surface markers, and enzymes [11]. A novel therapeutic strategy is the editing of macrophage (re)polarization. It is becoming more widely acknowledged that metabolic cascades are traits and regulators of macrophage activation. While M2 macrophages rely on OXPHOS for long-term energy production, M1 cells use glycolysis for quick death. These metabolic cascades directly determine the phenotype in addition to reflecting the energy production of macrophages. As a result, OXPHOS promotes M2 activation while glycolysis propels inflammatory macrophage responses. In other words, it can be argued that macrophage polarization is closely linked to changes in glycolytic and OXPHOS metabolism. According to immunometabolic theory, pro-inflammatory M1 macrophages mostly use glycolysis, whereas pro-reparative M2 macrophages use OXPHOS that is powered by fatty acids [12, 13]. Glycolysis is increased, mitochondrial OXPHOS is decreased, and pro-inflammatory cytokine production is raised when the principal macrophage glucose transporter, GLUT1, is overexpressed. Similarly, macrophages that lack GLUT1 exhibit higher M2 polarization [12]. Glycolysis is reduced and an M2 phenotype is encouraged by the knockdown of pyruvate dehydrogenase kinase 1, which phosphorylates pyruvate dehydrogenase to prevent its function in the production of pyruvate-derived acetyl CoA. Macrophage metabolic changes are necessary for the control of pro- and anti-inflammatory signalling pathways in addition to energy demands. In a positive feedback system, metabolic changes are also influenced by pro- or anti-inflammatory signals. Glycolysis can be initiated rapidly and produces ATP more rapidly than OXPHOS, although in significantly lower quantities. This is crucial for prompt macrophage activation and responses during infection or wound healing. Additionally, M1 macrophages’ need for glycolysis supports a number of important pro-inflammatory macrophage processes. To adjust to hypoxic conditions, the first and most evident change is a switch to anaerobic metabolism. The pentose phosphate pathway is more activated when glycolysis is upregulated. This raises NADPH levels for NADPH-oxidase to produce antimicrobial ROS and lipid synthesis, which promotes the expression of pro-inflammatory genes. Antioxidants like glutathione, which buffer excessive ROS generation during inflammation, are also biosynthesized using NADPH. M1 macrophages’ glycolytic shift is accompanied by a shortened tricarboxylic acid cycle (TCA) in the mitochondria, where succinate buildup encourages the stabilization of HIF-1α and the generation interleukin-1β (IL-1β). Furthermore, glycolytic enzymes can have significant signalling functions. For instance, pyruvate kinase M2 also serves as a nuclear HIF-1α co-activator, and glyceraldehyde-3-phosphate dehydrogenase is a part of the gamma-interferon-activated inhibitor of translation complex, which can prevent the translation of a number of inflammatory mRNAs. On the other hand, anti-inflammatory M2 macrophage actions are regulated by activation of mitochondrial OXPHOS, which is primarily driven by β-oxidation of fatty acids [14]. By interrupting glycolysis, the TCA cycle is restored by increasing pyruvate entry into the mitochondria; therefore, succinate accumulation is reduced and, consequently, HIF-1α activation. When the TCA cycle is restored, more NADH and FADH2 enter the ETC (electron transport chain), which raises OXPHOS. Alpha-ketoglutarate, a co-factor for the epigenetic activation of M2 genes, is also produced in greater amounts when the TCA cycle is intact. Furthermore, the expression of uncoupling protein 2 (UCP2) is increased in M2 macrophages. UCP2 uncouples OXPHOS and reduces the excessive production of mitochondrial ROS, which are responsible for increased M1 gene expression. Despite the above, glycolytic M1 macrophages also require the activity of complex I of the ETC, not to produce ATP but to generate ROS [12, 15]. Similarly, under certain conditions, M2 macrophages can also continue to utilize glycolysis in addition to OXPHOS. Additionally, distinct metabolic responses are mediated by various pro-inflammatory stimuli, and immunometabolic profiles vary among disease states. In summary, it has been demonstrated that M1 activation impairs mitochondrial activity, compromising subsequent IL-4 responses [11]. Specifically, it has been determined that the factor inhibiting M1→M2 repolarization is inhibition of M1-associated OXPHOS. M2 macrophages, on the other hand, are more plastic and can easily repolarize when in an M1-inflammatory state. To enhance metabolic and phenotypic reprogramming to M2 macrophages, inhibiting the generation of nitric oxide, a crucial effector molecule in M1 cells, reduces the deterioration in mitochondrial function. Therefore, OXPHOS is blunted by inflammatory macrophage activity, which stops repolarization. In order to adjust to the metabolic requirements of the cell, mitochondria, which are also a component of a communication network, regularly change their morphology [16]. The competing processes of fission (division) and fusion (joining) govern the dynamics of mitochondria. The two outer mitochondrial membrane (OMM)-bound proteins, mitochondrial fission factor and mitochondrial fission 1, as well as cytosolic dynamin-related protein 1, are the main players in fission. Inner membrane-bound optic atrophy 1 (Opa1) and outer membrane-bound mitofusin 1/2 play a major role in controlling fusion. Mitochondria fuse and fission to meet the cell’s evolving metabolic demands, which have significant effects on the fate and function of the cell. Thus, it has been postulated that the pro-inflammatory and pro-resolving phases of macrophage function are directly influenced by mitochondrial fission and fusion. Susser et al. discovered in a recent study that mitochondrial length directly influences the behaviour of macrophages, particularly when they shift from a pro-inflammatory to a pro-resolving state [16]. Consequently, improving the reprogramming of inflammatory macrophages into anti-inflammatory cells to manage illnesses may be possible through the therapeutic restoration of mitochondrial activity.

From mitochondrial dysfunction to mtDNA release: an interconnected mode of work

Many mitochondrial components can act as DAMPs when released into the cytosol and be recognized by specific receptors, pattern recognition receptors or cytoplasmic sensors [17], contributing to host defense and promoting sterile inflammatory responses [18]. Mitochondria have been shown to play a direct role in activating the immune response, as several mitochondrial products, when released from mitochondria, can directly trigger an innate immune response. Among these, we recognize human mtDNA, which encodes 13 subunits of the mitochondrial electron transport chain and ATP synthase, providing the wiring for the OXPHOS system [19, 20], and encodes 22 tRNAs and 2 rRNAs, essential for mRNA translation in the mitochondrial matrix [21]. mtDNA lacks histones and effective repair mechanisms, and therefore, mtDNA may be more prone to stress-induced damage [22]. Consequently, stress conditions promote mtDNA damage and then the release from mitochondria to the cytosol or extracellular space. When released into the cytosol under various cellular stress conditions, it acts as a DAMP, inducing innate immune and inflammatory responses by activating neutrophils and endothelial cells [23, 24].

mtDNA release can be triggered by several factors, including microbial infections, inflammation, gene mutations or deletions, and mitochondrial stress [25, 26]. To reach the cytosol, mtDNA must cross the inner mitochondrial membrane (IMM) primarily through pores such as the mitochondrial permeability transition pore (mPTP), a nonspecific mitochondrial channel activated by Ca²⁺ influx [27, 28], or by herniation of the IMM through the OMM. Passage through the OMM can be mediated by oligomerization of Bcl-2-associated X protein (BAX) and the Bcl-2 antagonist/killer homologue (BAK) [29]. Furthermore, under conditions of oxidative stress, the voltage-dependent anion channel 1 (VDAC1) can oligomerize and mediate mtDNA release. VDAC-mediated mtDNA release, but not BAX/BAK oligomerization, has been shown to be involved in numerous pathological conditions. However, the OMM pore that mediates mtDNA release depends on the level of mitochondrial stress: VDAC1 oligomerizes for moderate-level stress and BAX/BAK macropores for extreme-level stress and/or apoptosis [30] (Fig. 1).

Fig. 1.

Mechanisms of mtDNA release. mtDNA can be released from mitochondria into the cytosol and/or the extracellular environment through the BAK/BAX pore, the mPTP pore, and the GSDMD pore. Following apoptotic stimulation, the BAX/BAK pore releases intact ox-mtDNA into the cytosol. Due to Ca²⁺ overload and overproduction of ROS, the mPTP opens and the VDAC oligomerizes. Simultaneously, intact ox-mtDNA formed within the mitochondria is fragmented and released through the mPTP. Furthermore, GSDMD, cleaved by Caspase-1/4/5/8/11, forms GSDMD-NT fragments, which form the GSDMD pore, facilitating the release of ox-mtDNA fragments. Under various stress conditions, the formation of MDVs, herniation of the IMM through the outer membrane (single-membrane MDVs), or the genesis of double-membrane MDVs leads to the incorporation of mitochondrial contents, including mtDNA. Fusion of MDVs with the multivesicular body allows their release into the extracellular space

During specific cell death pathways, mtDNA can be released into the cytosol. During apoptosis, the imbalance between anti- and pro-apoptotic proteins of the Bcl-2 family causes activation of the pro-apoptotic BAX and BAK proteins, with translocation of cytosolic BAX into the OMM where it oligomerizes with BAK, forming pores through which mitochondrial components are released into the cytosol. This mechanism results in activation of the apoptosome, a protein complex composed of APAF1, mitochondrial cytochrome c, and inactivated caspase-9, that drives cell death by caspases-3/7 activation [31]. Although it is known that the programmed cell death process of apoptosis does not induce inflammatory processes, mediated for example by the action of caspases 3 and 7 which inhibit cGAMP synthase (cGAS) and the transcription interferon regulatory factor 3 (IRF3) of the pro-inflammatory cGAS-STING pathway, in pro-inflammatory apoptosis sustained by caspase deficiency, mtDNA accumulates in the cytosol supporting a subsequent initiation of the inflammatory process [32–34]. In this process, the IMM extrudes through the BAX/BAK pores on the OMM and permeabilizes, allowing the release of mtDNA into the cytosol [16, 17], where pro-inflammatory pathways are activated through interaction with cytosolic sensors that, in caspase-3/7 deficiency, are not subject to proteolytic cleavage, thus, triggering the inflammatory cascade is possible [35].

mtDNA is highly susceptible to oxidative damage, particularly to the formation of 8-oxo-2’-deoxyguanosine (8-oxo-dG) induced by ROS generated during mitochondrial respiration. Under physiological conditions, this lesion is removed via the base excision repair pathway, in which the enzyme OGG1 recognizes and removes 8-oxo-dG, and FEN1 processes repair intermediates such as DNA flaps to maintain genomic integrity [36, 37]. However, under chronic stress conditions or when the repair system is inefficient, oxidative damage persists, and FEN1 can contribute to the cleavage of damaged mtDNA, generating oxidized fragments of approximately 500–650 bp [28]. These fragments can escape from the mitochondrion through the mPTP and VDAC and accumulate in the cytoplasm to act as DAMPs. Increased mitochondrial OGG1 stability has been associated with reduced cytosolic release of oxidized mtDNA and mitigation of inflammation in several pathological conditions [38, 39].

Under conditions of mitochondrial stress, mPTP opening can mediate mtDNA fragments release in addition to the release of cytochrome c and other apoptotic factors through the IMM [40, 41]. In fact, pharmacological blockade of mPTP with cyclosporine A significantly reduces mtDNA release in nucleus pulposus cells, indicating its decisive role in mtDNA loss [42]. BAX/BAK macropores of the OMM, unlike the mPTP, allow the passage of large molecules, leading to IMM extrusion and mitochondrial herniation, resulting in the release of mtDNA into the cytoplasm. This release can occur independently of mPTP opening, through a process known as mitochondrial inner membrane permeabilization. Additionally, the presence of mtDNA in the cytoplasm can, in turn, induce BAX and BAK transcription, creating a vicious cycle that amplifies mitochondrial damage. Furthermore, BAK forms smaller pores more rapidly than BAX, leading to rapid mtDNA release, while simultaneously promoting BAX oligomerization to assemble larger pores, accelerating sustained mtDNA escape [43]. However, given the role of BAX and BAK, it is logical to think that their modulators may intervene indirectly on mtDNA release.

Among these, we recognize phosphoglycerate mutase family member 5, which initiates mtDNA release by dephosphorylating BAX, thus facilitating its recruitment to the mitochondrial membrane [44], as well as serine/arginine-rich splicing factor 6 (SRSF6), which regulates BAX alternative splicing, preventing excessive cell death. Knockout of SRSF6 leads to the accumulation of the BAX-κ variant and induces mtDNA escape [45]. Regulation of VDAC oligomerization on the OMM also indirectly influences mtDNA release, as Ca²⁺ in the endoplasmic reticulum promotes oligomerization and consequently mtDNA release [46].

When the IMM herniates through the VDAC1 oligomeric pores, it can form mitochondrial-derived vesicles (MDVs) [47] (Fig. 1) that transport mtDNA, along with other mitochondrial components, to lysosomes or reach the extracellular space through their release through the multivesicular body along with exosomes and extracellular vesicles [48, 49]. However, recent studies demonstrate that these vesicles exhibit variability in their membrane composition, existing as single-membrane structures derived from the inner or OMM or as double-membrane vesicles. It is important to underline that the biogenesis of MDVs and their composition is determined by the metabolic state of the cell, mediated by specific protein structures and stimulated by specific metabolites, such as fumarate, which, when pathologically accumulated, starts a cascade that leads to the biogenesis of MDVs rich in mtDNA correlated to an inflammatory state [50].

During pyroptosis, the processing of gasdermin D (GSDMD) also contributes to the release of extracellular mtDNA. Pyroptosis, triggered by pathogens or disruption of cellular homeostasis, is characterized by the inflammasome-dependent activation of caspase-1 or the non-canonical activation of murine caspase-11 or human caspase-4/-5. These caspases perform proteolytic cleavages on GSDMD, forming the amino-terminal GSDMD (GSDMD^NT), which binds to the plasma membrane and generates oligomeric pores responsible for plasma membrane permeabilization. During apoptosis, caspase-3 mediates the same process on GSDME, another member of the gasdermin protein family, forming GSDME^NT. Although plasma membrane permeabilization is more studied, cellular organelle membranes, such as OMM, have recently been described as targets of these proteins. Evidence shows that OMM permeabilization is induced independently of GSDMD activation and plasma membrane pore formation, but GSDMD is recognized as a critical factor in the severe fragmentation of the mitochondrial network after caspase-1 activation and therefore serves as an important factor for the release of mtDNA into the cytosol [51].

Maintenance of mitochondrial cristae is also essential for preserving mtDNA release. In Alzheimer’s disease models, reduced expression of the mitochondrial protein Opa1 contributes to the disease, but its overexpression helps reduce mtDNA loss in neurons, suggesting a potential therapeutic effect against mitochondrial inflammation. SAM50 deficiency compromises mitochondrial cristae structure, promoting mtDNA leakage into the cytoplasm through the formation of BAX/BAK pores. Loss of PHB1 in macrophages and Mitofilin also impairs cristae integrity and activates mPTP and VDAC channels, facilitating mtDNA leakage and triggering inflammation [46].

Activation of inflammatory pathways by cytosolic mtDNA

The major mitochondrial DAMP that has emerged so far to contribute to the systemic inflammatory response is mtDNA. It can elicit various proinflammatory signalling pathways via activation of cGAS and STING, the Nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome, or toll-like receptor 9 (TLR9), depending of the location [52].

Cytosolic mtDNA is recognized by cGAS, a protein localized in the cytosol and nucleus [53]. Nuclear cGAS is tightly controlled to prevent an unwanted inflammatory response [54], while binding of dsDNA in the cytosol frees the catalytic site of cGAS, allowing its activation. It has a canonical structure of members of the cGAS/DncV-like nucleotidyltransferase family: a cage-like architecture creates a deep pocket where the enzymatic site is located. cGAS binds cytosolic DNA, forming an oligomeric complex, where multiple copies of cGAS bind DNA to form a protein-DNA ladder [55]. DNA-bending and U-turning proteins, such as TFAM, preorganize DNA into the correct position to form multimers [56]. DNA binding causes cGAS to catalyze the production of cGAMP, a second messenger, which binds STING. Active dimeric STING moves from the endoplasmic reticulum to the Golgi compartment, recruits the kinase TBK1, which subsequently phosphorylates STING at its C-terminal tail. Phosphorylated STING recruits and allows phosphorylation of IRF3 by TBK1 [57]. Phosphorylated IRF3 dimerizes and translocates to the nucleus to drive transcription of type I interferon [58]. STING also positively regulates inflammatory cytokines and chemokines by activating the kinase IKK, which phosphorylates and inactivates the IkB family of inhibitors of the transcription factor NF-kB, which, like IRF3, translocates to the nucleus [33, 35, 59, 60].

mtDNA, particularly when oxidized under conditions of oxidative stress, when released into the cytosol can bind NLRP3 to trigger inflammasome assembly [28, 61]. NLRP3 is a large cytosolic complex, whose activation involves two phases: “priming” and “activation”. In the priming phase, receptors such as TLRs bind to their ligands, promoting the nuclear translocation of NF-κB to induce the transcriptional expression of NLRP3, IL-1β, and interleukin-18 (IL-18). In the activation phase, NLRP3 oligomerizes in response to various endogenous and exogenous stimuli. It is composed of a NLR, an adaptor apoptosis-associated speck-like protein containing a caspase activation and recruitment domain [CARD] (ASC), the protein kinase NIMA-related kinase 7, and a caspase-1 precursor protein. NLR recognizes DAMPs, such as mtDNA, and undergoes conformational changes that allow it to recruit and activate ASC by interacting with the N-terminal pyrin domain of NLR. ASC uses CARD to recruit pro-caspase-1. Caspase-1 then initiates inflammatory cascades, triggering its self-cleavage and releasing its active p20 fragment, which converts pro-IL-1β and pro-IL-18 into their mature, pro-inflammatory forms, producing the inflammatory cytokines, IL-1β and IL-18 [62, 63]. Furthermore, activated caspase-1 can cleave GSDMD to form the GSDMD^NT pore on the plasma membrane, thereby secreting IL-1β and IL-18 into the extracellular environment [64, 65].

mtDNA released into the cytosol or extracellular space can activate TLR9 signaling which results in the activation of several proinflammatory cytokines [66–68]. TLRs play an important role in regulating innate immunity and inflammation. Among several TLRs, endosomal/extracellular TLR9 is the only receptor for sensing unmethylated cytosine-phosphate-guanine (CpG)-rich DNA, which binds in a sequence-specific manner to the N-terminus of the C-shaped leucine-rich repeat region of TLR9 [69]. Binding of DNA molecules to each monomer results in TLR9 dimerization [70] and subsequent interaction with the myeloid differentiation primary response 88 (MYD88) adaptor protein [71]. The adaptor recruits the kinases IRAK to form the MyD88, IRAK4, IRAK1, and IRAK2 complex, which promote the sequential recruitment of TRAF6, TAK1, and MAPK, leading to the activation of the transcription factors NF-κB, AP-1, and CREB to produce inflammatory cytokines. Alternatively, MyD88, IRAK4, and IRAK1 promote the sequential recruitment of TRAF6 and TRAF3, leading to the activation of IRF7 and the production of IFN [60, 72] (Fig. 2).

Fig. 2.

Innate immune signaling driven by mtDNA. Cytosolic mtDNA binds to cGAS receptors, TLR9, and NLRP3 to trigger innate immune responses. (a) Cytosolic mtDNA can be recognized by cGAS, thereby promoting the production of cGAMP, which binds to and activates STING, thereby inducing the translocation of IRF3 and/or NF-κB to the nucleus to increase the expression of IFN-I, IL-6, and TNF. (b) Cytosolic NLRP3 recognizes and binds to ox-mtDNA to form the NLRP3 inflammasome with ASC and pro-caspase-1, inducing the maturation of pro-caspase-1, which cleaves pro-IL-1β and pro-IL-18 to produce mature forms of IL-1β and IL-18. (c) TLR9 translocates to the endosome membrane where it recognizes unmethylated CpG in mtDNA and subsequently activates MyD88, promoting the recruitment of IRAK4/IRAK1 or IRAK4/IRAK2. Through the assembly of TRAF 6/3 or TRAF6, TAK1, and MAPK, the translocation of IRF7 and the NF-κB, AP1, and CREB complex into the nucleus is promoted, increasing the expression of interferon and pro-inflammatory cytokines, respectively

Ultimately, mtDNA acts as a potent DAMP that activates several key inflammatory pathways through integrated mechanisms, making it a key mediator of the systemic inflammatory response.

Metabolic messengers in metabolic rewiring of immune cell metabolism

As mentioned above, mitochondrial function is a fundamental regulator of immune cell activation and subsequent function. In quiescent immune cells, OXPHOS provides the basal bioenergetic support necessary to maintain cellular quiescence. Upon activation, immune cells undergo a fundamental metabolic shift from OXPHOS to aerobic glycolysis [73]. This process, which is mechanistically and phenotypically similar to the Warburg effect in malignant cells, not only facilitates the rapid generation of ATP but also supplies the necessary biosynthetic intermediates to support cellular anabolism in proliferation, cytokine synthesis, and specialized effector functions. Oxidative mitochondrial metabolism might not appear important in affecting the fate of immune cells during inflammation [74]. However, under mitochondrial dysfunction conditions that could show ROS production, the immune responses are influenced by redox signalling. Thus, it arises that mitochondrial integrity and metabolic flexibility are indispensable for orchestrating effective immune responses [75].

Key immunometabolic signal is linked to metabolic reprogramming with immune regulation [76]. Cell fate and function under stress and inflammation can be reshaped by itaconate, a metabolic intermediate with origins in the TCA. Itaconate is synthetized by the enzyme aconitate decarboxylase 1, encoded by immune-response gene 1 (IRG1), during the transformation of citrate to isocitrate via the intermediate substrate cis-aconitate [77, 78]. The switch of metabolism is rewritten by itaconate, inhibiting the succinate dehydrogenase (SDH) [79]. SDH activity is known to be responsible in some pathological situations to improve the oxidative stress necessary to trigger the inflammatory process [80], promoting reverse electron transport (RET) in mitochondrial complex I, generating ROS. However, in mitochondria, the substrate succinate accumulates together with other metabolites of the TCA cycle, shifting this mitochondrial metabolic pathway from an energy-producing mode to sustain cataplerotic pathways providing precursors for biosynthesis, rather than energy production [81]. Moreover, IRG1 gene expression is positively regulated by IRFs presence in cells in response to pro-inflammatory processes [82]. The related increase in itaconate synthesis will contribute to reducing the inflammation by blocking RET and mitochondrial RNA release for the inflammatory signalling cascade through SDH inhibition [83]. Itaconate also helps to regulate immune cell states by altering macrophage polarisation, which supports anti-inflammatory responses and improves the local immunological microenvironment. Indeed, macrophage polarisation is critical to inflammation regulation [84], and itaconate supports M2 polarisation by inhibiting glycolytic enzymes. As a consequence, glycolytic flux is modulated accordingly, balancing inflammatory output and oxidative stress [76].

A recent study suggests that glucocorticoids (GCs) exert anti-inflammatory effects by regulating mitochondrial metabolism in macrophages, resulting in increased synthesis of itaconate [85]. The anti-inflammatory actions of glucocorticoids involve reprogramming macrophage mitochondrial metabolism, which inhibits the inflammatory response. In M1 polarized macrophages treated with lipopolysaccharide (LPS), the GCs can reverse the LPS-induced block of mitochondrial respiration and the process of LPS-induced aerobic glycolysis. As a result, GCs treatment of LPS-activated macrophages stimulates a highly energetic phenotype typical of M2 polarization. In the mechanism of the anti-inflammatory mode of action of GCs, the hormones and GCs receptors interact with parts of the pyruvate dehydrogenase complex present in the cytosol. It is plausible that the cytosolic GCs receptor contributes to a non-genomic anti-inflammatory response by facilitating the translocation of cytosolic pyruvate dehydrogenase to mitochondria through an alternative pathway involved in GCs-mediated anti-inflammatory signalling [75]. The result is a stimulation of flux of the TCA cycle in otherwise pro-inflammatory macrophages. Thus, the TCA cycle in pro-inflammatory macrophages is increased, rewiring of mitochondrial metabolism potentiated by TCA-cycle-dependent metabolites production such as citrate, fumarate and malate, as well as within the TCA-cycle-derived metabolite itaconate [85]. Substantially, GCs promote the influx of pyruvate into the mitochondrial oxidative metabolism (TCA cycle and OXPHOS) and rescue of mitochondrial respiration; on the contrary, GCs reduce the cytosolic use of pyruvate in lactate production.

Immune-metabolic disorders: mitochondria’s role

Both the regulation of health and the advancement of disease depend heavily on mitochondria. In particular, mitochondrial dysfunctions have been linked to a number of prevalent diseases, such as cancer, metabolic syndrome, neurodegeneration, and cardiovascular disorders [1].

Below are some examples of pathologies involving innate immunity and mitochondrial dysfunction. After an acute myocardial infarction (MI), innate immune cells are crucial for tissue damage and recovery [5]. Myeloid-specific deletion (mKO) of the mitochondrial complex I protein, which is encoded by Ndufs4, was shown to replicate the proinflammatory metabolic profile in macrophages and enhance the response to LPS in research by Cai et al. [5]. Thirty days following MI, mKO mice displayed reduced cardiac function, poor scar formation, and higher mortality. A longer shift to the repair phase, higher cell death of infiltrating macrophages, and an elevated inflammatory response were noted within 7 days following MI. Additionally, mKO macrophages exhibited poor efferocytosis, decreased expression of tissue repair factors and anti-inflammatory cytokines, and suppressed myofibroblast activation and proliferation in the infarcted area. mtROS scavenging improved myofibroblast function in vivo, corrected these deficits, and reduced post-MI mortality in mKO animals. All of these findings point to the crucial role mitochondria play in tissue healing and inflammation resolution through efferocytosis modulation and fibroblast interaction [5].

Another example is myocardial ischemia-reperfusion injury, where immune disorders play a major role in disease progression by exacerbating cardiac cell damage and mitochondria-related metabolic abnormalities [6]. In a study conducted by Cheng et al., low-immunogenic therapeutic strategies were used to improve the compromised immuno-metabolic microenvironment. Specifically, a syngenic reparative macrophage system was developed for the selective delivery of nanoscale drugs [6]. The release of nanodrugs into the ischemic myocardial region synergistically promoted cardiac cell survival and activated extracellular repair and angiogenesis, thus exerting long-term cardioprotective effects. Specifically, STING-related signaling pathways were inhibited, thus reshaping immuno-inflammatory homeostasis, increasing M2 macrophages, reparative cardiac resident macrophages, and regulatory T cells, and reducing the recruitment and infiltration of M1 macrophages and neutrophils. Additionally, mitochondrial oxidative phosphorylation was promoted, and mitochondrial-associated ferroptosis and oxidative damage were reduced [6].

In 2020, Deo et al. reported how innate immune cells use mitochondrial health monitoring to detect infections [7]. They demonstrate how macrophages exposed to outer membrane vesicles (OMVs) from P. aeruginosa (Pseudomonas aeruginosa), uropathogenic E. coli (Escherichia coli), and N. gonorrhoeae (Neisseria gonorrhoeae) trigger NLRP3 inflammasome activation and mitochondrial apoptosis. The unstable BCL-2 family member MCL-1 is depleted and BAK-dependent mitochondrial apoptosis is induced when OMVs and toxins that cause mitochondrial malfunction restrict host protein synthesis. Following OMV exposure in vitro, the NLRP3 inflammasome is activated by potassium ion efflux and mitochondrial apoptosis. Crucially, in the in vivo context, mitochondrial apoptosis controls the activation and release of interleukin-1β in response to N. gonorrhoeaeOMVs [7].

In cancer, the cGAS/STING pathway is a key immune activator, triggering innate immunosurveillance and responding to endogenous mtDNA [8]. Since serine is necessary for cellular metabolism and affects tumor growth and immunological responses, it has been demonstrated that serine deprivation has a major effect on the cGAS/STING pathway. In fact, serine deficiency in cells led to mitochondrial malfunction and the cytosolic release of mtDNA, which triggered type I IFN responses and activated the cGAS/STING pathway. Serine deprivation improved anticancer immunity in mice models by increasing tumoral immunological infiltration, which included type I IFN responses and CD4⁺/CD8⁺ T cells [8]. Clinically, immune activation and better survival were associated with a genetic profile in colorectal cancer patients that indicated decreased serine enrichment. Moreover, serine depletion improved the effectiveness of immune checkpoint inhibition, as evidenced by the considerable tumor volume reduction and long-term immunity in mice that resulted from combining serine deprivation with PD1 blockage [8].

Other pathologies, in which the main pathophysiological process is represented by the innate immune response, are disorders of the ocular surface exposed to multiple environmental stresses, such as dry eye diseases [41]. According to Ouyang et al., in these pathologies, mtDNA is released into the cytoplasm through the mPTP under stress, which further triggered the cGAS/STING pathway and exacerbated ocular surface damage and subsequent inflammatory reactions. Inflammatory reactions are decreased by genetic deletion, pharmacological suppression of STING, and blockage of mtDNA release [41].

The intricate role of mtDNA in shaping the immune microenvironment represents a critical frontier in immunometabolism research. The release of mtDNA into the cytosol, acting as DAMP, initiates inflammatory responses, notably the switch polarization of macrophages from M1 to M2 phenotypes. However, the precise molecular mechanisms governing this phenotypic shift remain largely unresolved. Future research must definitively establish the pathways through which mtDNA-mediated signalling influences macrophage plasticity. Open therapeutic questions include the pharmacologic tuning of STING signalling, a key sensor of cytosolic mtDNA, and the potential for metabolic checkpoint control to interrupt the M1/M2 axis. Furthermore, a deep understanding of methods for the therapeutic modulation of mtDNA release is essential to develop novel strategies for manipulating the innate immune system in chronic inflammation and cancer.

In immunometabolic disorders and tissue damage, mitochondrial components such as mtDNA, mitochondria-located microRNA, and related proteins may be used as therapeutic agents to improve mitochondrial function [1] (Fig. 3).

Fig. 3.

Mechanisms through which mtDNA as a mitochondrial DAMPs activate innate immune responses. Three main pathways are highlighted: cGAS-STING, NLRP3, and TLR9 which, through specific intracellular signals, induce the production of inflammatory cytokines and type I interferons, activating the innate immune response. These processes are implicated in the pathogenesis of several diseases, including cardiovascular disease, neurodegeneration, metabolic syndrome, cancer, and dry eye diseases. The final section proposes therapeutic strategies aimed at modulating mtDNA release and the activation of inflammatory pathways

Conclusions

On balance, we highlight a pivotal regulatory node within the immunometabolic network, underscoring the essential interplay between mitochondrial integrity, DAMP-mediated signalling, and innate immune cell programming. We try to suggest the mitochondrial components, particularly mtDNA, as critical danger signals whose cytosolic release orchestrates inflammatory reprogramming, a mechanism central to the persistence of chronic sterile inflammation.

However, despite advances in understanding the role of mitochondrial dysfunction in immunometabolism, numerous questions remain that deserve further exploration. In particular, therapeutic modulation of mtDNA release represents a promising frontier for attenuating the aberrant activation of inflammatory pathways. Targeted interventions on mitochondrial pores (such as mPTP, VDAC1, BAX/BAK) could limit the dispersion of mtDNA into the cytosol, reducing the initiation of sterile immune responses. In parallel, pharmacological regulation of the cGAS-STING pathway, for example through selective STING inhibitors or cGAMP modulators, offers new possibilities for controlling the expression of interferons and proinflammatory cytokines. Finally, controlling metabolic checkpoints such as GLUT1 and UCP2 could allow reprogramming of immune metabolism, favoring polarization toward pro-resolving M2 phenotypes. The integration of these strategies could pave the way for innovative therapies for chronic inflammatory, autoimmune, and degenerative diseases, with a significant impact on regenerative medicine and immunotherapy.

Abbreviations

IFNγ

Interferonγ

TLR

Toll-like receptor

LPS

Lipopolysaccharide

OXPHOS

Oxidative phosphorylation

ROS

Reactive oxygen species

TCA

Tricarboxylic acid cycle

ETC

Electron transport chain

UCP2

Uncoupling protein 2

Opa1

Optic atrophy 1

DAMPs

Damage-associated molecular patterns

mtDNA

Mitochondrial DNA

IMM

Inner mitochondrial membrane

mPTP

Mitochondrial permeability transition pore

OMM

Outer mitochondrial membrane

BAX

Bcl-2-associated X protein

BAK

Bcl-2 antagonist/killer homologue

VDAC1

Voltage-dependent anion channel 1

IRF3

Interferon regulatory factor 3

8-oxo-dG

8-oxo-2’-deoxyguanosine

SRSF6

Serine/arginine-rich splicing factor 6

MDVs

Mitochondrial-derived vesicles

GSDMD

Gasdermin D

GSDMD^NT

Amino-terminal GSDMD

cGAMP

2′3′-cyclic GMP-AMP

cGAS

Cyclic GMP-AMP (cGAMP) synthase

STING

Stimulator of interferon genes

NLRP3

NLR family pyrin domain containing 3

TLR9

Toll-like receptor 9

IL-1β

Interleukin-1β

IL-18

Interleukin-18

NLR

Nod-like receptor

CARD

Caspase activation and recruitment domain

ASC

Adaptor apoptosis-associated speck-like protein containing a CARD

MYD88

Myeloid differentiation primary response 88

IRG1

Encoded by immune-response gene 1

SDH

Succinate dehydrogenase

RET

Reverse electron transport

GCs

Glucocorticoids

MI

Myocardial infarction

mKO

Myeloid-specific deletion

OMVs

Outer membrane vesicles

Author contributions

Cristina Algieri contributed to drafted and revised the manuscript; Salvatore Nesci contributed to the conception, to drafted and revised the manuscript; Francesca Oppedisano contributed to drafted and revised the manuscript.

Funding

Not applicable.

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors serve on the editorial board of JTRM: CA is Associate Editor, SN is the section editor of Cellular Metabolism Therapy; FO is Associate Editor.