Mitochondrial Damage-Associated Molecular Patterns and Metabolism in the Regulation of Innate Immunity

Abstract

The innate immune system, as the host’s first line of defense against intruders, plays a critical role in recognizing, identifying, and reacting to a wide range of microbial intruders. There is increasing evidence that mitochondrial stress is a major initiator of innate immune responses. When mitochondria’s integrity is disrupted or dysfunction occurs, the mitochondria’s contents are released into the cytosol. These contents, like reactive oxygen species, mitochondrial DNA, and double-stranded RNA, among others, act as damage-related molecular patterns (DAMPs) that can bind to multiple innate immune sensors, particularly pattern recognition receptors, thereby leading to inflammation. To avoid the production of DAMPs, in addition to safeguarding organelles integrity and functionality, mitochondria may activate mitophagy or apoptosis. Moreover, mitochondrial components and specific metabolic regulations modify properties of innate immune cells. These include macrophages, dendritic cells, innate lymphoid cells, and so on, in steady state or in stimulation that are involved in processes ranging from the tricarboxylic acid cycle to oxidative phosphorylation and fatty acid metabolism. Here we provide a brief summary of mitochondrial DAMPs’ initiated and potentiated inflammatory response in the innate immune system. We also provide insights into how the state of activation, differentiation, and functional polarization of innate immune cells can be influenced by alteration to the metabolic pathways in mitochondria.

Introduction

Mitochondria are descended from a common ancestral organelle originating from the integration of an endosymbiotic alpha-proteobacterium into a host cell related to Asgard Archaea [1]. Known as “powerhouses,” they are vital subcellular compartments that provide energy in the form of ATP through the electron transport chain (ETC, also called the respiratory chain) and oxidative phosphorylation (OXPHOS) [2]. The ETC comprises complexes I–IV, as well as the electron transporters ubiquinone (coenzyme Q [CoQ]) and cytochrome c, which, as a single unit, is coupled with complex V (ATP synthase) to carry out OXPHOS [3]. In mitochondria, electrons from reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) produced in the tricarboxylic acid (TCA) cycle donate electrons to complex I (NADH dehydrogenase) and complex II (succinate dehydrogenase), respectively, while CoQ and cytochrome c shuttle electrons to complex III (cytochrome c reductase) and complex IV (cytochrome c oxidase) [4]. The generated proton gradient is then exploited by complex V, which phosphorylates ADP into ATP [5].

Beyond providing energy, mitochondria are also highly immunogenic through potent innate immune pathways. Mitochondria are the only subcellular organelles in animals with a DNA genome other than the nucleus. Located in the mitochondrial matrix, mitochondrial DNA (mtDNA) is a double-stranded DNA (dsDNA) molecule structured in a loop without histones [6]. mtDNA encodes 37 genes, all of which are associated with OXPHOS and normal mitochondrial function [7]. Since mtDNA replicates independently from the nuclear genome, it exposes the organism’s prokaryotic ancestry and could be recognized by the immune system as “foreign”. These “foreign” substances are present in intracellular damage-associated molecular patterns (DAMPs), which primarily originate from damaged cells or dysfunctional mitochondria [8]. Pattern recognition receptors (PRRs) serve as primary cellular sensors for danger signals in the innate immune system. They recognize highly conserved molecular patterns that include exogenous “non-self” pathogen-associated molecular patterns (PAMPs) and intrinsic “host-self” DAMPs, resulting in an intracellular signaling cascade [9, 10]. If mtDNA is released from the mitochondria or exits the cell, it has the potential to be internalized by PRRs located in the cytoplasm or on the cell surface, subsequently triggering inflammatory mechanisms associated with DAMPs or immune cell activation [11–13].

Normally, a tiny number of electrons leaving the ETC interact with oxygen to generate reactive oxygen species (ROS); ROS are the primary source of free radicals and also are byproducts of mitochondrial metabolism [14]. Complex I has been regarded as the primary source of ROS. The formation of ROS including superoxide and hydrogen peroxide, in complex I can result from NADH oxidation, ubiquinone reduction, and the energy-consuming reverse electron transfer that causes NAD+ reduction and requires a high transmembrane electrical potential [15, 16]. Besides complex I, complexes II and III can also participate in ROS generation since their components can promote oxygen reduction, leading to superoxide anions (O₂^•-) [17]. Increasing evidence has shown that excessive ROS formation may cause OXPHOS dysfunction, DNA damage, protein oxidation, irreversible mitochondrial impairment, and, eventually, cell death [18, 19]. Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. The mechanism by which UCPs are activated appears to involve redox signaling through mild uncoupling and mitochondrial membrane depolarization that feedbacks to prevent robust ROS generation [20]. Furthermore, mitophagy, the selective autophagic degradation of dysfunctional mitochondria, can ultimately eliminate dysfunctional mitochondria to maintain normal physiological processes, is one of the quality control mechanisms in cells, and can protect cells from injury [21].

Herein, we discuss in detail the mitochondria-derived DAMPs, including mtDNA fragments, ATP, and ROS, released into the cytosol or the extracellular environment due to the loss of membrane integrity; this process further drives inflammatory mediator production and exacerbates the inflammatory response (shown in Fig. 1), as well as mitochondrial protective mechanisms. We also describe mitochondrial metabolism regulation in the presence of different innate immune cells at steady or inflammation.

Fig. 1.

Mitochondrial DAMPs activate innate immune pathways. mtDNA released into the cytosol as whole nucleoids through BAX–BAK macropores associated with apoptosis or as oxidized fragments through VDAC1 and mPTP is recognized by cGAS and signals through cGAMP and STING to activate inflammatory gene transcription. mtDNA leaked from a cell can be phagocytosed and bind endosomal TLR9, triggering MyD88-dependent signaling to interferons and proinflammatory cytokines. The NLRP3 inflammasome is activated by oxidized mtDNA from dysfunctional mitochondria. TFAM mainly regulate mtDNA transcription. Activation of the RLRs MDA5 and RIG-I by mtRNA is initiated in the cytosol, but signaling depends upon binding MAVS on the outer mitochondrial membrane to activate their downstream signaling pathways. ATP that leaks from a damaged cell can trigger NLRP3 inflammasome. Cardiolipin can mediate NLRP3 inflammasome and interact directly with LC3 in response to mitophagy process. Enhanced production of mtROS elevated oxidative stress levels lead to the leakage of DAMPs.

Mitochondria as Activators in PRRs

At present, PRRs can be divided into nucleotide-binding oligomerization domain-like receptors (NLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), which are intracellular molecules. They can also be divided into Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), both of which are localized to the cell or endosomal membranes. Mitochondria have indeed become critical activators of these PRRs, initiating innate responses necessary for pathogen elimination by activating corresponding PRRs (shown in Table 1).

Table 1.

Mitochondria as activators of pattern recognition receptors

PRRs	Reported roles in immune response	Reference
TLRs	Migration of mitochondria to macrophage phagosomes following the activation of certain TLRs (TLR1, TLR2, and TLR4) enhances mtROS generation in antibacterial response	[22–24]
	Pharmacological inhibition of mtROS effectively reduces LPS-induced MAPK phosphorylation and inflammatory cytokine production in cells from individuals with TRAPS	[25]
	mtROS was the primary contributor for TLR4-induced ROS generation in gastric cancer patient cells; the blocking of mtROS production resulted in cell cycle arrest	[26]
	Poly(I-C) activates TLR3, which prompts the mitochondrial apoptotic pathway	[27]
NLRs	Mitochondrial ATP and certain bacterial toxins have the ability to cause NLRP3 inflammasome activation	[28]
	Mitochondrial coordination of NLRP3 localization is implicated in regulating NLRP3 activity	[29]
	Dysfunctional mitochondria inhibit the activation of inflammasome through producing mtROS	[30, 31]
	FAO deficiency in mitochondria can activate NLRP3 inflammasomes in the absence of CPT1A	[32]
	NLRX1 can regulate mitochondrial antiviral immunity through N-terminal mitochondrial targeting sequence, and also can regulate the production of mtROS, thereby reducing oxidative stress and apoptosis in tissue damage	[33]
RLRs	Mitochondria prompt RLRs to activate RNA species and provide the optimum signaling adapter MAVS required for antiviral signal transduction	[34–36]

TLRs, Toll-like receptors; NLRs, nucleotide-binding oligomerization domain-like receptors; RLRs, retinoic acid-inducible gene I-like receptors; mtROS, mitochondrial ROS; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinases; TRAPS, tumor necrosis factor receptor-associated periodic syndrome; NLRP3, NLR family pyrin domain-containing 3; FAO, fatty acid oxidation; CPT1A, carnitine palmitoyltransferase1A; MAVS, mitochondrial antiviral signaling protein.

TLRs are among the most well-studied and well-characterized PRRs. Mitochondria are intimately involved in the regulation of TLR activities. It has been observed that the migration of mitochondria to macrophage phagosomes following the activation of certain TLRs (TLR1, TLR2, and TLR4) enhances mtROS generation in antibacterial response [22]. The tumor necrosis factor receptor-associated factor 6 (TRAF6) – a TLR signaling adapter – is translocated to the mitochondria as part of this response, and it interacts with the protein evolutionarily conserved signaling intermediate in toll pathway (ECSIT) simultaneously [23]. The ubiquitination and enrichment of ECSIT at the mitochondrial periphery increase mitochondrial and cellular ROS production via interaction with TRAF6 [22, 24]. Furthermore, Bulua et al. [25] discovered that pharmacological inhibition of mitochondrial ROS effectively reduces lipopolysaccharide (LPS)-induced mitogen-activated protein kinases’ (MAPK) phosphorylation and inflammatory cytokine production in cells from individuals with tumor necrosis factor receptor-associated periodic syndrome (TRAPS), an autosomal dominant autoinflammatory disease with TNFRSF1A gene variation. Yuan et al. [26] showed that mtROS was the primary contributor for TLR4-induced ROS generation in gastric cancer patient cells; mtROS production blocking by diphenylene iodonium (DPI) resulted in cell cycle arrest and the loss of mitochondrial potential. Additionally, according to one study, poly(I-C) activates TLR3, which prompts the mitochondrial apoptotic pathway [27].

NLRs are intracellular molecules belonging to the PRR family. Among them, the NLR family pyrin domain-containing 3 (NLRP3) inflammasome is a well-studied member in the host defense response, which consists of the NLRP3 scaffold, the apoptosis-associated speck-like protein adapter (ASC), and caspase-1 [30, 37]. NLRP3 inflammasomes mediate caspase-1 activation, which leads to the maturation of interleukin 1β (IL-1β), and subsequently induces cell death (pyroptosis) under pathological inflammation and stress [30, 38]. Mounting evidence points to dysfunctional mitochondria emerging as key activators of NLRP3 inflammasomes. Initially, it was shown that mitochondrial ATP and certain bacterial toxins have the ability to cause NLRP3 inflammasome activation [28]. Subsequently, mitochondrial coordination of NLRP3 localization is implicated in regulating NLRP3 activity [29]. Inflammasome’s activation is inhibited when dysregulating mitochondrial activity by voltage-dependent anion channel (VDAC) inhibition through mtROS [30, 31]. Furthermore, fatty acid oxidation (FAO) deficiency in mitochondria can activate NLRP3 inflammasomes in the absence of carnitine palmitoyltransferase 1A (CPT1A), a key rate-limiting enzyme of FAO [32]. Additionally, NLRX1 regulates mitochondrial antiviral immunity with a mitochondria-targeting sequence in the N-terminus, which modulates virus-induced interferon β (IFNβ) production. NLRX1 could also potentially trigger the generation of mtROS, reducing oxidative stress and apoptosis in tissue injury [33].

RLRs triggered by mitochondria are indispensable for the generation of IFN and proinflammatory cytokines in response to viral RNA [39]. The RLR family includes three receptors – RIG-I, MDA5, and DHX58 – all of which can sense single- or double-stranded RNA (dsRNA). When they recognize RNA, RIG-I and MDA5 become activated, interact with each other, and initiate a downstream mitochondrial antiviral signaling protein (MAVS) [34]. The formation of RIG-I/MAVS multimers on the surface of mitochondria then recruits signaling molecules such as TRAF3 and TRAF6, thereby promoting antiviral immunity [35, 36]. As such, mitochondria play a central role in RLR signaling pathways. First, mitochondria prompt RLRs to activate RNA species following a mitochondrial imbalance; second, mitochondria act as a physical platform, providing the optimum signaling adapter MAVS required for antiviral signal transduction [36, 40].

Mitochondrial Damage-Associated Molecular Patterns in Innate Immune Response

Mechanism of mtDNA Leakage

Mitochondria exhibit high structural plasticity through continuously orchestrated fission and fusion to remodel their morphology and cellular location [41, 42]. This process, called mitochondrial dynamics, is how they control their own functions and the cell’s metabolism. The combination of two or more damaged mitochondria is part of mitochondrial fusion, “functionally complementing” the harmful effects of mitochondria with damaged components or mutated mtDNA [43, 44]. The mitochondrial membrane is made up of two different membranes that are distinct in function: outer mitochondrial membranes (OMM) and inner mitochondrial membranes (IMM) [45]. Members of the dynamin GTPase family include the fusion proteins mitofusin 1 (Mfn 1), Mfn 2, and optic atrophy 1 (OPA1), all of which mediate the processes of OMM and IMM fusion, respectively [46]. Mitochondrial fission, mediated by dynamin-related protein 1 (Drp1) and Fission 1 (Fis1), involves the division of mitochondria into smaller pieces [47]. This is essential for the production of sufficient mitochondria in developing cells, the energy metabolism, the spread and inheritance of mitochondria, and the removal of damaged or dysfunctional mitochondria via mitophagy [48–51]. Fission also facilitates apoptosis through the release of cytochrome c, playing a crucial role in this process [52, 53].

When oxidative damage in the mitochondria accumulates, mtDNA can be released into the cytosol in the form of circular structures or DNA fragments, where the mtDNA becomes a vital driver of inflammation [54]. The opening of the mitochondrial permeability transition pore (mPTP) is the driving process that leads to mtDNA leakage [13, 55]. Stable protein-permeable pores are formed from the oligomerization of the pro-apoptotic proteins BCL-2-like protein 4 (BAX) and BCL-2 homologous antagonist/killer (BAK) on the OMM. These pores facilitate not only mitochondrial herniation of the IMM and ejection of cytoplasmic content such as mtDNA, but also the leakage of cytochrome c, thereby triggering the intrinsic apoptotic pathway [56–58]. Mild mitochondrial stress results in the release of mtDNA fragments rather than whole nucleoids, and this process is mediated via the permeability transition pore complex (PTPC) on the IMM. Along similar lines, the release of mtDNA fragments into the cytoplasm is facilitated by ROS-induced oligomerization of the VDAC1 on the OMM. Current research suggests that the presence of circulating mtDNA can modulate the production of proinflammatory cytokines in humans with chronic inflammation [59]. The crucial pathway of mtDNA release into extracellular space is mediated by plasma cell ruptures triggered by cell necrosis or passive apoptosis, or by a pathway that actively releases extracellular vesicles to elicit apoptosis.

mtDNA Trigger Pattern Recognition Receptors Signaling

Among the mitochondrial DAMPs, mtDNA is currently emphasized in our bodies due to unmethylated CpG and formylated peptides [60]. mtDNA release into the cytoplasm and extracellular environment mediates the activation of at least three PRRs and innate immune responses, including cyclic GMP-AMP synthetase, interferon gene (cGAS-STING) pathway stimulation, TLR9, and NLRP3 inflammasome formation [12, 61, 62].

Activated cGAS-STING signaling has garnered attention in particular for stimulating innate immune defense programs since it is principally responsible for recognizing foreign DNA [11]. cGAS, known as a cytosolic DNA sensor, forms an oligomeric complex with DNA and undergoes switch-like conformational changes. cGAS then uses ATP and GTP as substrates to produce 2′,3′-cyclic GMP-AMP (cGAMP), which is a second messenger molecule and potent agonist of STING [63–65]. cGAMP activates STING, an endoplasmic reticulum (ER)-residing adapter that undergoes ER-to-Golgi trafficking and tetramer formation via a higher order oligomerization [66]. Upon activation, STING translocates to intermediate compartments between the ER and the Golgi apparatus; it drives the transphosphorylation of TANK-binding kinase 1 (TBK1), and it phosphorylates interferon regulatory factor 3 (IRF3) to trigger the production of ISGs and type I interferons after entering the nucleus [67]. STING activation prompts the translocation of NF-kB to the nucleus and subsequent gene transcription activation that produces proinflammatory cytokines like IL-6 and tumor necrosis factor, recruiting IB kinase (IKK), and thereby phosphorylating NF-kB inhibitor IκBα. Additionally, the cGAS-STING signaling activates other cellular processes, including LC3-mediated autophagy, apoptosis, and necroptosis [58].

TLR9 is an intracellular PRR that can sense DNA in cells’ immune complexes [68]. When unmethylated CpG-rich DNA sequences stimulate TLR9-dependent responses, TLR9 translocates from the ER to the endosomal membrane. In the presence of mtDNA, TLR9 is directly activated and associated with MyD88 through its TIR domain, recruiting the IL-1 receptor-associated kinase (IRAK) and several downstream pathways, including NF-kB, to provoke an innate response when in combination [69]. Many studies implicate the role of mtDNA in TLR9-mediated pathogenesis in the host body. In research by Bao and colleagues, mtDNA encouraged tumor-associated macrophage (TAM) recruitment and polarization through the TLR9-mediated NF-κB signaling pathway in hepatocellular carcinoma [70]. Additionally, TLR9, upon detecting mtDNA release from necrotic cells, triggers ERK1/2-dependent signaling, leading to vascular contractility [71]. Oxidative mtDNA released by neutrophils also drives IFN production via the TLR9 pathway in human systemic lupus erythematosus (SLE) [72].

NLRP3 inflammation is present primarily in innate immune cells, including macrophages, monocytes, dendritic cells (DCs), and neutrophils, whose main function is to regulate caspase-1-dependent formally programmed apoptosis as well as proinflammatory cell death. Previous studies have shown that classical NLRP3-activating stimulation leads to mtROS generation and mtDNA release. Recent evidence suggests that the synthesis of mtDNA is essential for NLRP3 signaling priming [73]. Enhanced mtROS production mediates cytosolic mtDNA translocation to an oxidized form (Ox-mtDNA), proposed to bind cytosolic NLRP3, thereby triggering inflammasome assembly and activation [74, 75]. According to the study of Li et al. [76], the NLRP3 inflammasome is bound and activated by induced mtDNA oxidation and release, resulting in antiviral inflammation responses.

mtRNA Coordinate Innate Immune Signaling

mtDNA transcription produces overlapping products that can form double-stranded RNA called mt-dsRNA [77]. Recently, transcribed mtRNA have been found in separate clusters closer to mitochondrial nucleoids. Elevated oxidative stress levels lead to the leakage of mtRNA into the cytoplasm [78, 79]. Zhou et al. [80] showed that cytoplasmic mt-dsRNA can be sensed by MDA5 (melanoma differentiation-associated protein 5, a member of the RLR family), which induces NF-κB activation. Additionally, the binding of mt-dsRNA triggers RIG-I to localize into the mitochondria and interact with the MAVS protein, thereby coordinating the activation of IFN-inducing pathways and autophagy [81, 82].

ATP

Although ATP is a ubiquitous energy source, it also acts as a mitochondrial DAMP, primarily produced in the mitochondria by ATP synthase connected to the IMM’s ETC in cells. When mitochondrial homeostasis is compromised, ATP functions as a DAMP following ATP’s release from cells through vesicular exocytosis and extracellular processes such as ATP release channels [83]. NLRP3 activation requires ATP, which is capable of altering the structure of NLRP3 [84]. Furthermore, extracellular ATP acts on the purinergic P2 × 7 receptor and induces K+ expulsion from the cell, resulting in caspase-1 cleavage [85].

mtROS

Most ROS are generated in the mitochondria. mtROS produced upon the leakage of electrons in impaired mitochondria leads to mitochondrial membrane potential loss, oxidative damage, and redox-sensitive signaling pathway activation. For instance, mtROS is critical for the expression of the central mitochondria-associated adapter MAVS and enhances RLR activity in the regulation of the innate host defenses against viruses [82, 86]. mtROS enhances autophagy by activating the phosphatidylinositol 3 kinase (PI3K)/AKT/mTOR cascade, increasing autophagic signaling via phosphorylating ULK1, and upregulating Atg proteins, which are crucial regulators of autophagy and mitophagy [87]. mtROS bursts result in increased TRAF6 levels contributing to this pyroptosis [88]. Since mtDNA encodes for crucial OXPHOS, its subunits are widely known. Damage to mtDNA caused by ROS (Ox-mtDNA) contributes to OXPHOS deficiencies, which in turn contribute to cellular damage by failing to meet cells’ energy needs [89]. Moreover, mtROS is one of the main mediators of NLRP3 inflammasomes [90].

Other Damage-Associated Molecular Patterns

Mitochondrial transcription factor A (TFAM) is a nuclear-encoded protein that plays a significant role in mtDNA transcription, packaging, and maintenance [91]. The immunological regulation of mitochondria by TFAM is complex. TFAM concentrations are proportional to the number of mtDNA copies [92]. Reduced TFAM protein levels change the way mtDNA is packed, causing the release of fragments into the cytosol, where they activate the cGAS. Furthermore, a lack of TFAM increases mtROS and apoptosis via altering the mitochondrial membrane permeability [93]. However, mtDNA can be distorted by the attachment of TFAM, resulting in mtDNA internalization and enhanced TLR9 detection. Zhang et al. [94] discovered that TFAM silencing and genetic ablation stopped the activation of the NLRP3 inflammasome through mtDNA leakage.

Cardiolipin is a mitochondrial-exclusive phospholipid and is fundamental to the structure and function of mitochondrial membranes [95]. Detaching from the IMM and externalizing on the OMM, cardiolipin can interact directly with LC3 in response to pro-mitophagy stimuli [96]. Additionally, the localization and activation of NLRP3 inflammasomes can be mediated by cardiolipin [97]. A recent study discovered that the translocation of IL-1 precursor to the mitochondria, where it directly interacts with mitochondrial cardiolipin, both interrupted cardiolipin-LC3b-dependent mitophagy as well as increased Nlrp3 inflammasome activation and IL-1 production in LPS priming of macrophages [98]. Cardiolipin interacts with cytochrome c, promoting ROS-mediated apoptosis [99]. Extracellular unsaturated cardiolipin can act as a TLR4 inhibitor by preventing LPS binding and TLR4 dimerization [100].

Regulation of Mitochondrial Damage-Associated Molecular Patterns Signaling

To minimize inflammatory reactions triggered by mitochondrial DAMPs and to maintain a healthy mitochondrial population, mitochondria evolved two mechanisms: autophagy and apoptosis (shown in Fig. 2). Mitophagy is a cellular process for the degradation of mitochondria by autophagic machinery, acting as the primary mechanism for mitochondrial quality control [101]. Misfunctioning mitochondria in various pathological processes, such as oxidative stress and inflammation, can lead to the depolarization of the IMM and reduce the cleavage of the protein PTEN-induced kinase 1 (PINK1) at the OMM [102]. Accumulated PINK1 is autophosphorylated and activated, recruiting Parkin, a cytosolic E3-ubiquitin ligase [102, 103]. Parkin subsequently ubiquitinates mitochondrial proteins such as VDAC1, Mfn1, and Mfn2, leading to their degradation. This in turn induces mitochondrial fission and mitophagy [104, 105]. Ubiquitin is responsible for inducing the formation of phagophores in close proximity to mitochondria via optineurin (OPTN), a well-known cargo autophagy receptor [106]. Moreover, OPTN anchors Ub-labeled mitochondria into autophagosomes by directly connecting with their LC3. The OMM-localized mitophagy receptor BNIP3 (BCL-2 interacting protein 3) also targets mitochondria to mitophagy by interacting directly with LC3 during hypoxia, FUNDC1 (FUN14 domain-containing 1), and NIX (NIP3-like protein X) [107–109]. Failure in clearing defective mitochondria via mitophagy causes mitochondrial contents to leak into the cytosol when mitochondria are under stress. This stops the release of DAMPs and the activation of cytosolic PRRs [110]. Aside from mitophagy, damaged mitochondria can also be removed from cells through programmed cell death pathways, particularly apoptosis [111].

Fig. 2.

Regulatory mechanisms prevent the activation of innate immunity by mitochondrial DAMPs. Upon mitochondrial damage, mitochondria utilize PINK1/Parkin-dependent ubiquitin-driven mitophagy or mitophagy receptor-mediated mitophagy, and apoptotic pathway to minimize mitochondrial DAMP leakages and maintenance of a healthy mitochondrial population.

The mitochondrial intrinsic pathway of apoptosis is triggered when cells are subjected to extreme amounts of stress (such as substantial DNA damage, ER and replication stress, and continuous ROS bursts) [112–114]. Endogenous apoptosis is a mitochondrial-activated apoptosis process regulated by the Bcl-2 protein family [115]. Upon an apoptotic stimulus, the mitochondrial translocation of BAX proteins and activation of BAK proteins promote mitochondrial outer membrane permeability (MOMP), which increases pro-apoptotic factors such as cytochrome c and SMAC/DIABLO released from the mitochondria into the cytosol. This mechanism potentially activates the caspase cascade and ultimately leads to cell death [116].

So far, we have discussed that mitochondria serve innate immune purposes by providing immunogenic DAMPs and activating the corresponding PRRs. It cannot be ignored that the reprogramming of cellular metabolism of the innate immune cells has emerged as a major aspect of cell activation involved in innate immunity. Notably, mitochondria are also the centers of energy and metabolism in cellular organisms and are vital for cellular life, death, and differentiation.

Mitochondrial Metabolism in Innate Immune Cells

Immune Cell Metabolism in Mitochondria

A healthy and intact mitochondrial metabolism, as the primary energy producer in immune cells, is critical for the maintenance of cellular activity [117]. Mitochondria generate ATP for cellular processes by OXPHOS via the ETC and the TCA cycle [117, 118]. The TCA cycle refuels NADH and FADH to generate substrates for the ETC. Multiple carbon substrates undergo catabolism to feed into the TCA cycle [119], such as the metabolism of glucose by glycolysis. After uptake, glucose undergoes intracellular processes to become pyruvate, which can be converted into not only acetyl-CoA and CO₂ by pyruvate dehydrogenase (PDH) and NAD+/NADH but also oxaloacetate by PDH in mitochondria. Both acetyl-CoA and oxaloacetate are substrates of TCA. In addition to glucose, fatty acids and glutamine can drive the TCA cycle. Fatty acids provide an effective mechanism for the production of acetyl-CoA through FAO. Glutamine is converted to derived alpha-ketoglutarate via glutaminolysis for later utilization in the TCA cycle [120].

Activation and Functional Changes in Immune Cells

Metabolic changes in immune cells are crucial for a successful immune response during steady state, activation, or inflammation [121]. Changes in metabolism of the various innate immune cells (mainly including macrophages, DCs, and ILC monocytes) will be described in more detail below (shown in Fig. 3).

Fig. 3.

Mitochondrial metabolism in resting and activated innate immune cells. M1 cells mainly use glycolysis and glutaminolysis upon inflammation, displaying a dysregulated TCA cycle and increased rate of lipid synthesis meet the highly biosynthetic and bioenergetic demand, whereas M2 macrophages rely on mitochondrial OXPHOS and fatty acid oxidation for sustained energy production. M1 macrophages are hardly repolarized into an M2 phenotype upon IL-4 exposure due to suppression of mitochondrial OXPHOS. NK cells preferentially utilize glucose, which is metabolized by glycolysis to fuel OXPHOS and ATP production. ILC2s preferentially generate energy by breaking down FAs, but not glucose, for OXPHOS. Arg1 is required for ILC2 cytokine production and optimal proliferation and promotes ILC2 glycolytic capacity. pDCs produce type 1 IFNs for activation due to increased glutaminolysis and OXPHOS, while reduced OXPHOS activity was observed in CD1c+ myeloid DCs.

Macrophages

Macrophages are significant innate immune cells that strategically position throughout the body tissues [122]. Under the influence of various stimulating substances, macrophages have high plasticity and can be classified into two groups: classic macrophages (M1), for immune surveillance against viral infections and an anti-tumor effect, and alternatively activated macrophages (M2), which are immunomodulatory and control tissue remodeling [123]. In response to IFN-γ, LPS, and granulocyte-macrophage colony-stimulating factors (GM-CSF), M1 macrophages upregulate molecules of major histocompatibility complex II (MHC-II) and enhance the production of numerous inflammatory mediators, such as IL-6, ROS, and NO [123, 124]. M2 macrophages express activation markers, including CD68, CD163, and CD206, and they secrete TGFβ, IL-10, and arginase stimulated by IL-4 or IL-13 [123, 125].

A balance between the macrophages’ various active states plays a crucial role in immunity homeostasis. It has recently become recognized that mitochondrial health and metabolic status are extremely important for determining macrophage phenotype [126]. M1 and M2 macrophages have distinct regulation mitochondrial metabolism. A shift from mitochondrial oxidative metabolism to glycolysis induces M1 proinflammatory polarization [127].

LPS stimulation promotes glucose absorption and glycolysis in macrophages. It has been discovered that a shift in the expression of 6-phosphofructo-2-kinase (PFK2) to the more active ubiquitous PFK2 isoenzyme (which enhances glycolytic rate via accumulating fructose-2,6-bisphosphate (F2, 6BP) in macrophages [128]) results from the deep mechanism of a HIF-1a dependence mode. Researchers discovered that M1 macrophages are hardly repolarized into an M2 phenotype upon IL-4 exposure in vitro and in vivo [129]. M1-associated suppression of mitochondrial OXPHOS is identified as the mechanism impeding M1 to M2 repolarization [129]. Another typical feature of proinflammatory macrophage activation is that two crucial enzymatic processes in the reprogramming of TCA are down-regulated in LPS-stimulated M1 macrophages, thereby inhibiting the TCA cycle. On the one hand, the downward adjustment of isocitrate dehydrogenase (IDH) results in citrate buildup, which permits citrate carriers on the mitochondrial membrane to transport citrate to the cytoplasm for the synthesis of FA, prostaglandin (PG), and NO. On the other hand, succinate dehydrogenase (SDH) is down-regulated, leading to the accumulation of succinate and inducing the expression of IL-1β in a HIF-1α-dependent manner.

As opposed to M1 macrophages, M2 cells with extended mitochondria derive their energy primarily from FAO and OXPHOS [130]. Glycolysis is impaired in M2 macrophages due to low HIF-1α activity and uPFK2. Furthermore, increased glucose use for UDP-GlcNAc production has been proposed as a metabolic marker of the M2 phenotype [131]. In the process of M2 macrophage polarization, IL-4 stimulation induces the transcription of peroxisome proliferator-activated receptor (PPAR)γ and PPARδ [132]. These play a dominant role in adipogenesis and the storage of FAs as triglycerides (TAG) in adipocytes [133]. Arginine metabolism and glutamine metabolism exert a favorable influence on M2 polarization [134, 135]. Notably, mitophagy plays an important role in reprogramming macrophages metabolically. The mitophagy deficiency weakens cell glycolysis, increases cell OXPHOS, further increases macrophage M2 polarization, and ultimately promotes tumor growth during the IL-4-induced polarization process [136].

Dendritic Cells

DCs, known as quintessential antigen-presenting cells (APCs), are responsible for bridging the gap between innate and adaptive immunity. DCs are often divided into two groups: plasmacytoid (pDCs) and conventional DCs (cDCs). As in macrophages, the activation and function of DCs rely on the rapid induction of glycolysis, which is required for TLR signaling [137]. Upon TLR stimulation, human blood DC subsets undergo distinct mitochondrial remodeling and metabolic adaptation. In response to the TLR9 agonist CpGA, pDCs produce type 1 IFNs due to increased glutaminolysis and OXPHOS [138], which can be prevented by the inhibition of glutaminolysis and OXPHOS. While reduced OXPHOS activity was observed in CD1c+ myeloid DCs, BNIP3-dependent mitophagy is required for CD1c+ myeloid DC activation and glycoly induction [139]. Notably, the consequences of fatty acid production vary significantly between DCs isolated from tumors and DCs that are TLR activated [140].

Innate Lymphocytic Cells

Innate lymphoid cells (ILCs) are mainly tissue-resident cells with rapid activation by microenvironmental stimuli [141]. Localized preferentially in mucosal barriers, they play a role in monitoring and maintaining innate immunity. Three different ILC groups can be distinguished based on signature surface markers, genes expressed, and effector cytokines, all of which parallel the function of adaptive T cell subset counterparts similar to CD4+ T helper lymphocytes: type 1 ILCs, type 2 ILCs (ILC2s), and type 3 ILCs (ILC3s) [142]. Group 1 ILCs, including ILC1 and natural killer (NK) cells, primarily protect the host from viruses or bacterial infection [143, 144]. ILC2s are commonly described as having a contradictory nature, as they are responsible for helminth immunity and tissue repair but also for pathogenic responses driving allergic inflammation [145–148]. ILC3s are implicated in gastrointestinal immune responses to fungal and bacterial pathogens.

Distinct mitochondrial metabolic profiles drive the functional fate of NK cells, which rely primarily on glucose for energy in a steady state [149, 150]. Upon activation, cytotoxic NK cells promote cytotoxicity by increasing glycolysis and OXPHOS rates. Under hypoxic and glycolytic limitations, NK cells are polarized to a regulatory state with low levels of glycolysis and OXPHOS and may utilize other fuels such as fatty acids or amino acids. In other studies, mitophagy plays a specific role in memory NK cell formation following viral infection in a BNIP3-dependent manner [151, 152].

ILC2s have been shown to predominantly generate energy by FAO, which shows the highest level of FA uptake among the ILC subsets [153]. According to the outcomes of lipid tracing investigations, tissue-resident ILC2s enhance exogenous FA uptake and are transiently stored in lipid droplets (LD), preventing the disruption of mitochondrial membrane integrity [154, 155]. Autophagy is extensively used by activated ILC2s to maintain their homeostasis and effector function [156]. Arginase 1 (Arg1) is a metalloenzyme that catalyzes the hydrolysis of arginine to ornithine and urea; a novel source of Arg1 is ILC2s in resting tissue during allergic inflammation [157]. A mouse experiment has shown that ILC-intrinsic Arg1 deletion prevents type 2 lung inflammation by inhibiting ILC2 proliferation and decreasing cytokine production [157]. Mechanistically, this is accomplished by changing arginine catabolism, limiting polyamine production, and decreasing aerobic glycolysis. Similarly, in ILC2 activated by IL-33, an increased Arg1 enzyme level enhances the production of IL-5 and IL-13 through promoting the cell’s glycolytic capacity [158]. It becomes obvious that ILC2_10s demonstrate a metabolic dependency on the glycolytic pathway for IL-10 production, shifting from the FAO pathway conventionally utilized for immunoregulatory potential [159, 160].

ILC3s increase mitochondrial oxygen consumption as well as glucose and fatty acid metabolism in cell activation [161]. Researchers discovered that ILC3s proliferation and the generation of IL-22 and IL-17A depend on the mTORC1 and HIF1 pathway, which reprograms ILC3s metabolism toward glycolysis and maintains RORγt expression [162]. Additionally, mtROS stabilizes HIF1 and RORγt in ILC3s, promoting their activation [161].

Conclusion

Published evidence has clearly stated that mitochondrial-derived DAMPs are associated with innate immune signaling pathways via binding to certain PRRs. Unlike other organelles, mitochondria contain their own genome known as mtDNA, which encodes OXPHOS genes and in turn influences mitochondrial function and cell viability. Due to its bacterial DNA characteristics, it can be recognized by PRRs and provoke inflammation. However, further study is necessary to understand how mtDNA transcriptional machinery is regulated. Also needed is a more precise description of physiological mtDNA damage or mutations in chronic inflammation like aging and in neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. Interestingly, ROS is not only a mitochondrial DAMP in innate signaling pathways; ROS over-production could cause cellular oxidative stress, which regulates other DAMPs’ oxidation, such as ox-mtDNA. Functional and proper regulation of mitophagy and apoptosis are essential for cellular homeostasis and survival in innate responses.

Additionally, the impact of mitochondrial metabolism alteration is likely to be equally dependent on the metabolic demands of the particular cell type as well as the developmental stage of innate immune cells in pathogenic or inflammatory signals. Moreover, further research is needed regarding the role of mitochondrial immunometabolism in the generation and leakage of DAMPs and the subsequent inflammation in innate immune responses.

Conflict of Interest Statement

The authors have no conflicts of interest to declare in relation to this work.

Funding Sources

This work was supported by grants from the Shandong Provincial Natural Science Foundation, China (ZR2022LZL006) and the Science Foundation of Shandong First Medical University & Shandong Academy of Medical Sciences for Youth Program (202201-017).

Author Contributions

Yanmin Lyu and Tianyu Wang drafted and made the figures. Shuhong Huang and Zhaoqiang Zhang reviewed the manuscript.

Funding Statement

This work was supported by grants from the Shandong Provincial Natural Science Foundation, China (ZR2022LZL006) and the Science Foundation of Shandong First Medical University & Shandong Academy of Medical Sciences for Youth Program (202201-017).