Mitochondrial control of innate immune responses

Mitochondria are considered as critical signal platforms to facilitate the RLRs-MAVS signaling cascade by mediating the prion-like aggregation of MAVS (32) (Figure 2). Apart from nucleic acids-sensing TLRs located in endosomes (33), viral RNA can be recognized by RNA sensors in the cytoplasm (34). RIG-I (35) and melanoma differentiation-associated gene 5 (MDA5) (36), which belong to the DExD/H box RNA helicase family, are identified to detect cytosolic viral dsRNA. Upon viral dsRNA association, the conserved caspase activation and recruitment domains (CARDs) of these RNA sensors (37) are exposed and bind to the CARD domains of MAVS (38–41) located in the mitochondrial outer membrane, which drives the prion-like aggregation and activation of MAVS (32, 42). This aggregation recruits kinases TANK-binding kinase 1 (TBK1)/inhibitor of nuclear factor kappa-B kinase ϵ (IKKϵ) to activate the downstream transcriptional factors, interferon regulatory factor 3/7 (IRF3/7), and nuclear factor-κB (NF-κB). mRNAs of type I/III IFNs, interferon-stimulated genes (ISGs), and proinflammatory cytokines (43) are then transcribed and translated to restrict microbial infection, modulate adaptive immunity, and initialize tissue regeneration (44, 45). Intriguingly, the peroxisome (46, 47) and mitochondrial-associated endoplasmic reticulum membranes (MAM) (48) localization of MAVS and MAVS signalosomes have also been suggested, which induce or modulate the expression of ISGs and type III IFNs. Aberrant activation of RLRs-MAVS signaling is known to cause various autoimmune and autoinflammatory disorders (49).

Proteins that regulate mitochondrial dynamics (12), including the dynamin-related family of large GTPases mitofusin 1 (MFN1), mitofusin 2 (MFN2), optic atrophy 1 (OPA1), and dynamin-related protein 1 (DRP1), play critical roles in MAVS function. MFN1 facilitates the redistribution of MAVS on mitochondria to positively regulate RLR-MAVS signaling (50), while conversely, MFN2 represses MAVS aggregation (51). Additionally, both mitochondrial membrane potential (ΔΨ(m)) and membrane proteins can function as negative regulators of MAVS (7), such as NLR family member X1 (NLRX1), globular head domain of complement component C1q receptor (gC1qR), and Polo-like kinase 1 (PLK1). Activation of RLRs-MAVS signaling, in return, appears to contribute to the elongation of the mitochondrial network via interaction between MAVS and MFN1 (50, 52). Notably, disrupting the mitochondrial fragmentation function of DRP1 by TBK1-mediated S412 phosphorylation forms hyper-fused mitochondrial networks, which are required to effectively assemble large MAVS aggregates during innate RNA sensing (45). In this scenario, phosphorylation of DRP1 by TBK1 directly blocks the high-order oligomerization and mitochondrial division function of DRP1 (45). The TBK1-DRP1 axis also participates in nutrient-triggered mitochondrial dynamics and cell fate determination, suggesting that innate immunity also contributes to governing the morphology and physiology of mitochondria (45).

Inflammasomes are multi-subunit complexes activated under stress conditions to regulate inflammatory responses and induce pyroptotic cell death (53, 54). They consist of a receptor (NLR family and PYHIN protein family members), the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), and the inflammatory cysteine protease pro-caspase 1 (55). Mitochondria act as scaffolds for NLRP3 inflammasome assembly. NLRP3 translocates from the endoplasmic reticulum (ER) to mitochondria and the MAM to form the NLRP3 inflammasome, which leads to the maturation of caspase-1-dependent proinflammatory cytokines IL-1β and IL-18 (56, 57). Cardiolipin, MAVS, and MFN2 have also been shown to regulate NLRP3 inflammasome assembly. For instance, upon mitochondrial stresses, cardiolipin, the mitochondrial inner membrane-associated phospholipid, is exposed on the mitochondrial surface to serve as the independent binding site for NLRP3 and full-length caspase-1 to assemble and activate the inflammasome (58, 59). Alternatively, MAVS forms a complex with MFN2 (60) to recruit NLRP3 to the mitochondria for NLRP3 inflammasome assembly during RNA viral infection (61–63).

A recent study indicated the importance of mitochondrial signal platforms in telomere-mediated tumor suppression and aging. Z-conformation nucleic acid binding protein 1 (ZBP1) (64), an IFN-stimulated gene, functions as a cytosolic Z-nucleic acid sensor to regulate type I IFN signaling, inflammation, cell death, and tissue homeostasis (65). The study showed that mitochondria provide a scaffold for ZBP1-telomeric-repeat-containing RNAs (TERRA) complexes to activate MAVS-dependent interferon response during a replicative crisis (66).

These findings suggest that mitochondrial architecture, rather than a single mitochondria-related protein or product, is essential in maintaining various innate immune responses, which provide a broader perspective for studying the relationship between mitochondria, immunity, and diseases.

Mitophagy plays a crucial role in regulating type I IFN signaling activation. Deficient mitophagy caused by autophagy related 5 (ATG5) ablation increases mtROS production and elevates levels of MAVS, promoting the activation of the type I IFN pathway (72). Sequestosome 1 (SQSTM1/p62)-dependent mitophagy (73) and mitophagy induced by viral proteins (71, 74) also regulate the type I IFN response. By contrast, NIX-dependent mitophagy acts as an intrinsic negative regulator of the RLRs-MAVS axis by preventing spontaneous aggregation of endogenous MAVS in the absence of viral infection (75). Besides, the disruption of mitophagy caused by PINK1 and Parkin mutations contributes to the activation of the cGAS-STING signaling pathway via cytosolic accumulation of mtDNA (76), which will be elaborated on later.

It has been found that mitophagy helps to repress NLRP3 inflammasome activation by reducing mtROS production and mtDNA release via clearing damaged mitochondria (57, 67), while Parkin plays a crucial role in mtROS-NLRP3-mediated inflammatory response by regulating mitophagy activation (77). Defective mitophagy and mtROS accumulation induced by receptor-interacting protein kinase 2 (RIPK2) deletion lead to increased morbidity and mortality by accelerating IL-18 secretion and inflammatory activation during influenza A virus (IAV) infection (78). NF-κB signaling is also vital in anti-inflammatory by inducing the expression of SQSTM1/p62 to promote autophagic clearance of damaged mitochondria in lipopolysaccharide (LPS)-treated macrophages (79). Besides Ub-dependent mitophagy, FUNDC1-mediated Ub-independent mitophagy can control the secretion of inflammasome-related IL-1β (80). Additionally, mitophagy regulates the activation of AIM2 (absent in melanoma 2) (81) and NLRC4 (NLR-family CARD domain-containing protein 4) (82) inflammasomes, revealing a widespread role of mitophagy in controlling inflammasome-mediated innate immune responses.

Although a wide variety of studies have demonstrated the importance of cytosolic mtDNA in innate immunity, the mechanisms explaining the translocation of mtDNA from the mitochondrial matrix to the cytosol are still not well understood. In 2014, two independent studies uncovered that mtDNA is released during mitochondrial apoptosis (105, 106). Follow-up studies demonstrated that apoptosis-related MOMPs allow the rupture of the mitochondrial inner membrane and mtDNA release (107–110). Additionally, under subtle stress-induced non-apoptotic conditions, only a fraction of mitochondria undergoing permeabilization (minority MOMPs) also cause the cytosolic release of mtDNA and the activation of cGAS-STING signaling, the major pathway sensing of mtDNA in the cytosol (111, 112). Furthermore, VDAC, which is used for metabolites and ions transport (113), can oligomerize to form pores in the MOM under oxidative stress conditions (114) and interact with mtDNA and form oligomers in the MOM to permit the release of short mtDNA fragments and trigger type I IFN response (115), while MPTP, formed in the mitochondrial inner membrane under various cellular stress conditions, is considered to regulate the release of small mtDNA fragments (116, 117). Notably, cytoplasmic accumulation of TAR DNA binding protein 43 (TDP-43), a disease hallmark of amyotrophic lateral sclerosis (ALS), can trigger mtDNA release via the MPTP and VDAC1 oligomers to upregulate the NF-κB and cGAS-STING signaling (118). Meanwhile, the endonuclease Flap endonuclease 1 (FEN1) has been found to cleave oxidized mtDNA into 600-650 bp fragments that can be released from mitochondria via MPTP and VDAC-dependent channels (119). Although MOMP-induced mtDNA release in apoptotic cells has been discussed in detail, the regulations of mtDNA release in living cells under moderate-level stress and the role of the mitochondrial inner membrane in mtDNA release are still not well understood. These mechanisms of mtDNA release are summarized in Figure 3.

cGAS is a primary cytosolic DNA receptor that can recognize both exogenous and endogenous DNA and induce the production of 2’3’-cyclic GMP-AMP (cGAMP) (120, 121), which activates the adaptor protein STING (122–125) to elicit type I IFN and inflammatory responses (126). In addition to its role in response to viral and bacterial DNAs (127–131), the cGAS-STING pathway also elicits and controls self-DNA (nuclear DNA and mtDNA)-induced innate immune responses (132), inflammatory diseases (133, 134), antitumor immunity (135–137), neurodegenerative diseases (138), and diverse cellular functions (139) including protein synthesis (140) and glucose metabolism (141).

Various signals can disrupt mitochondrial integrity during pathogen infections. For instance, IAV triggers the release of mtDNA through the proton-selective ion channel (viroporin) activity of the M2 protein in a MAVS-dependent manner and evades the recognition of cGAS by interacting with NS1 proteins and mtDNA (142). Infections of herpes simplex virus 1/2 (HSV-1/HSV-2) have been found to cause stress and elimination of mtDNA, which regulates antiviral responses and ISG expression (143–146). Interestingly, infections with RNA viruses, such as Dengue virus, can activate the cGAS-STING (147–149) and TLR9 pathways (150) by causing mitochondrial stress and oxidized mtDNA release. Certain strains of Mycobacterium tuberculosis can also trigger cGAS-STING activation (128, 130, 151) by inducing mtDNA release (152). During pathogen infections, IL-1β secretion can upregulate antimicrobial immune responses by releasing mtDNA to activate the cGAS-STING signaling pathway (153), providing new insights into the mechanisms by which numerous cytokine-related pathways boost inflammatory responses. These results suggest that maintaining mtDNA homeostasis may be a beneficial regulator of innate antiviral immunity.

In addition to pathogen infection, the disruption of mitochondrial DNA integrity, replication, and repair can activate the cGAS-STING pathway (133). For instance, mitochondrial transcription factor A (TFAM) deficiency can cause cGAS-STING activation by damaging the nucleoid structure and accumulating cytosolic mtDNA (8, 145, 154), while depletion of sorting and assembly machinery component 50 (Sam50) (155) and endonuclease G (115) similarly induces mtDNA-related cGAS-STING activation in hepatocytes and other cells. Additionally, chemotherapeutic drugs (156), pyrimidine nucleotide carrier SLC25A33 overexpression, as well as the inhibition of de novo pyrimidine synthesis (157), can trigger mtDNA leaking and cGAS-STING activation. As a result, the importance of cGAS-STING in sensing mtDNA and inflammatory-related diseases has made it a highly targeted drug (158).

Nakahira et al. were the first to report that the MPTP-induced cytosolic accumulation of mtDNA and mtROS strengthens NLRP3 inflammasome activation in autophagic protein-deficient macrophages (159). mtDNA oxidized by mtROS is preferred for NLRP3 recognition (79, 160–162). Activating inflammasomes and caspases also regulate mtDNA release by causing mitochondrial damage. Notably, the role of NLRP3 may not promote mtDNA release but rather stabilize it in the cytoplasm (162), while other studies indicate that NLRP3 activation amplifies the mitochondrial damage and mtDNA release (163, 164). Caspase-1 activated by inflammasomes destroys mitochondria by triggering multiple pathways to promote the production of mtROS, the dissipation of ΔΨ(m), and the permeabilization of mitochondrial membranes (163). Moreover, NLRP3 promotes caspase-2 activation and BID cleavage during infection-related ER stress to facilitate mitochondrial permeabilization (164). Further studies are therefore needed to clarify those conflicting data and better understand the relationship between mtDNA and NLRP3.

TLR9 is a member of the TLRs family and is the first TLR discovered to sense bacterial DNA with hypomethylated CpG motifs, and this recognition activates the MAPK and NF-κB signaling pathways, leading to an inflammatory response (134, 165). Interestingly, TLR9 has also been found to recognize mtDNA released into the bloodstream during systemic inflammatory response syndrome (SIRS) and activate a p38-mediated inflammatory response (166, 167). mtDNA released from dying cells can form a complex with the antimicrobial peptide LL-37 to evade the degradation by DNase II and activate TLR9 response (168). In addition, the mtDNA-TFAM complex released from necrotic cells augments proinflammatory response by promoting the activation of receptor for advanced glycation end products (RAGE) and TLR9 (169, 170). Mitochondrial dynamics can also play a role in TLR9-induced inflammation by affecting mtDNA stability (171, 172). The absence of OPA1 in mice results in muscle atrophy and premature death due to the accumulation of damaged mitochondria and disruption of mitophagy, leading to mtDNA-related TLR9-mediated inflammation (173). Circulating mtDNA has been confirmed as an endogenous TLR9 agonist in various studies and has been implicated in several inflammatory-related diseases (9), including rheumatoid arthritis (22), atherosclerosis (168, 174), hypertension (175), acute liver injury (176) and non-alcoholic steatohepatitis (177).

mtDNA can also play a role in the extracellular space by engaging the cGAS-STING pathway and/or the TLR9 pathway on neighboring immune cells (24, 178, 179), such as in the scenario of neutrophil extracellular traps (NETs) during microbial infection and sterile inflammatory diseases (9, 180). NETs are vast extracellular decondensed-chromatin networks containing a plethora of microbial-killing proteins and DNAs (180, 181). In healthy neutrophils with oxidative damage, entire TFAM-mtDNA complexes are expelled into the extracellular space, and mtDNA can be transported into lysosomes to avoid recognition by TLR9 and maintain the immunological silence of plasmacytoid dendritic cells (pDCs) (179). Conversely, oxidized mtDNA is released in systemic lupus erythematosus (SLE) patients and activates inflammatory responses (179). The formation of oxidized mtDNA-containing NETs can be stimulated by ribonucleotide immune complexes (RNP-ICs) (178) and continuous IFN-α signaling (183), which further strengthens the cGAS-STING pathway. Additionally, lymphocytes and eosinophils can engage the type I IFN response in peripheral blood mononuclear cells by secreting mtDNA-containing webs (184, 185). These findings highlight the importance of mtDNA in both intracellular and extracellular pathways in regulating immune responses.

mtROS, the “so-called” byproducts of the mitochondrial respiration chain, is generated at complexes I and III of the ETC in response to hypoxia, substrate availability alteration, and abnormal mitochondrial conditions (193). mtROS has been demonstrated to play a crucial role in innate immunity (194) (Figure 4).

Glucose metabolism has been shown to suppress RLR-induced interferon production through lactate, which directly binds to MAVS and disrupts its mitochondrial localization (216). MAVS directly binds to hexokinase-2 (HK-2) in its resting state to maintain its kinase activity and proper glycolysis process (216). However, research has also shown that the cytosolic phospholipase A2 (cPLA2) disrupts the interaction between MAVS and HK-2 in astrocytes, leading to increased NF-κB-related inflammation (217) (Figure 5). Notably, a recent study revealed a critical role of AMPK in potentiating both RLRs-MAVS and cGAS-STING signaling and antiviral responses via direct AMPK-mediated phosphorylation of TBK1 at S511 residue (141). These mutual interactions between glucose metabolism and innate immunity indicate an intricate and delicate network of immune responses related to mitochondria.

Interplay of N-acetylglucosamine (GlcNAc) with hexokinase can lead to inflammatory responses in the host by disrupting hexokinase localization and activating NLRP3 inflammasomes (218) (Figure 5). Similarly, inhibiting glycolysis after the priming step through chemical treatment can activate the NLRP3 inflammasome (219). Free fatty acids (FAs) from diet or FA synthesis can activate the NLRP3 inflammasome (220–222). Therefore, activation of AMPK during fasting or caloric restriction suppresses FA-induced NLRP3 inflammasome activation by promoting autophagy and limiting ROS production (220, 223), in contrast to its role in potentiating nucleic acid signaling (141). Besides, in a state of low blood glucose, fatty acid oxidation provides energy and leads to the production of ketone bodies like β-hydroxybutyrate (BHB), which inhibit the activation of the NLRP3 inflammasome by inhibiting K⁺ efflux (224). By activating citrate synthase and inhibiting FA uptake, BHB reduces the level of mitochondrial acetylation, which represses NLRP3 inflammasome formation, mitochondrial dysfunction, and heart fibrosis (225). Butyrate, a short-chain fatty acid (SCFA), by contrast, inhibits NLRP3 activation by reducing pro-IL-1β levels (226).

Macrophages can be differentiated into two main distinct lineages on the type of activation signals they have received: M1 macrophages, characterized by a proinflammatory phenotype, and M2 macrophages, characterized by an anti-inflammatory and pro-fibrotic phenotype (227, 228). Studies have shown that oxygen consumption and reliance on mitochondrial metabolism differ between M1 and M2 macrophages (228–230). For example, M1 macrophages show reduced while M2 macrophages exhibit increased mitochondrial metabolism. Notably, the inhibition of the ECT through reverse electron transport (RET) increases the production of mtROS, stabilizes HIF-1α, and enhances the inflammatory response, favoring M1 macrophage polarization (228). Moreover, inhibiting fatty acid oxidation promotes M1 macrophage activation and suppresses M2 macrophage phenotypes (228).

Metabolites from the TCA cycle also play a vital role in controlling macrophage polarization by regulating macrophage chromatin modifications, DNA methylation, and protein post-translational modifications (231) (Figure 5). Acetyl-CoA, the starting point of the TCA cycle (231–235), provides acetyl groups for acetylation and influences the production of proinflammatory molecules such as nitric oxide (NO), ROS, and prostaglandin E2 (PGE2) in response to stimuli such as LPS (236). Acetyl-CoA in IL-4-related M2 macrophages, however, increases histone acetylation and M2 macrophage-related gene expression (237). The immune-responsive gene 1 protein (IRG1) produces itaconate (238, 239), which has been found to accumulate in response to Mycobacterium tuberculosis infection (182) and LPS stimulation (240) and exert anti-inflammatory effects (241). On the other hand, α-ketoglutarate (α-KG) promotes M2 polarization in LPS-stimulated M1 macrophages through epigenetic reprogramming and suppression of the NF-κB signaling pathway (242). Succinate similarly contributes to macrophage activation by stabilizing HIF-1α and increasing the transcription of IL-1β (243, 244), while fumarate accumulates in LPS-activated macrophages (243, 245) and modulates pro-inflammation by inhibiting lysine-specific histone demethylase 5 (KDM5) enzyme (246). These repeated observations indicate an essential role of mitochondrial metabolites in the polarization and activation of macrophages.