From powerhouse to modulator: regulating immune system responses through intracellular mitochondrial transfer

Abstract

Mitochondria are traditionally known as the cells' powerhouses; however, their roles go far beyond energy suppliers. They are involved in intracellular signaling and thus play a crucial role in shaping cells' destiny and functionality, including immune cells. Mitochondria can be actively exchanged between immune and non-immune cells via mechanisms such as nanotubes and extracellular vesicles. The mitochondria transfer from immune cells to different cells is associated with physiological and pathological processes, including inflammatory disorders, cardiovascular diseases, diabetes, and cancer. On the other hand, mitochondrial transfer from mesenchymal stem cells, bone marrow-derived stem cells, and adipocytes to immune cells significantly affects their functions. Mitochondrial transfer can prevent exhaustion/senescence in immune cells through intracellular signaling pathways and metabolic reprogramming. Thus, it is emerging as a promising therapeutic strategy for immune system diseases, especially those involving inflammation and autoimmune components. Transferring healthy mitochondria into damaged or dysfunctional cells can restore mitochondrial function, which is crucial for cellular energy production, immune regulation, and inflammation control. Also, mitochondrial transfer may enhance the potential of current therapeutic immune cell-based therapies such as CAR-T cell therapy.

Keywords: Immune system; Immunometabolism; Immunotherapy; Mitochondria; Mitochondria Transfer; Organelle therapy.

Fig. 1

The significance of mitochondria in intracellular signaling: RIG-I/MDA5 detection of viral RNA interacts with MAVS, resulting in the formation of MAVS signalosomes on the mitochondrial surface, which is necessary for recruiting TRAF proteins. TRAF proteins activate TBK1, IRF3/IRF7, and NF-κB, generating type I interferons. Additionally, mitochondria can provide a framework for inflammasome assembly through the interaction of activated NLRP3 with MAVS, hence increasing inflammasome formation and activity. Furthermore, MARCH 5 regulates TLR7 signaling by polyubiquitinating and degrading TANK. Following TLR activation, TRAF6 binds to and ubiquitinates ECSIT on the surface of mitochondria, causing mitochondrial migration to phagosomes and the production of mtROS. TLR4 activation activates NF-κB via assembling the TRAF6, ECSIT, and TAK1 complex, which leads to enhanced TAK1 kinase activity. TRAF; tumor necrosis factor receptor-associated factor, MARCH-5; mitochondrial protein membrane-associated ring-CH-type finger 5, TANK; TRAF family member-associated NF-kappa-B activator, ECSIT; evolutionarily conserved signaling intermediate in Toll

Fig. 2

Mitochondria enhance the phagocytic activity of neutrophils. Mitochondria in neutrophils contribute to various biological processes beyond apoptosis, including chemotaxis, ROS production, degranulation, and NET formation. As cytochrome c is released from the mitochondrial membrane, the apoptosome complex is formed, resulting in the subsequent activation of caspase 9 and caspase 3, consequently triggering apoptosis. MCU and the outer mitochondrial membrane protein MFN2 play key roles in neutrophil chemotaxis by regulating mitochondrial dynamics and maintaining interactions with the endoplasmic reticulum. Mitochondria can be an important source of intracellular ROS in neutrophils, and it is reported that mtROS is involved in the oxidative burst and the degranulation of neutrophils. Additionally, calcium release from the endoplasmic reticulum stimulates PAD4, enhancing histone citrullination and leading to chromatin decondensation (the starting point of NETosis). Both mtROS and mtDNA are important mediators of NET formation, assisting in releasing web-like structures consisting of chromatin and granule proteins like NE, MPO, and MMP-9, which trap pathogens. NETs serve an important role in pathogen capture and maintaining neutrophil performance. NET; neutrophil extracellular trap, MCU; mitochondrial calcium uptake transporter, PAD4, peptidyl arginine deiminase 4 NE; neutrophil elastase, MPO; myeloperoxidase, MMP-9; matrix metalloprotease 9

Fig. 3

Mitochondria play a crucial role in regulating both metabolic processes and immune responses in macrophages. Mitochondria play an important role in macrophage metabolism and immunological function, influencing their ability to respond to diverse immune stimuli via metabolic reprogramming. In M1 macrophages, glycolysis promotes pro-inflammatory cytokine production and increases bactericidal activity. This metabolic transition is connected with a disruption in the TCA cycle, resulting in the generation of metabolites such as citrate, which is then transformed into itaconate—a compound with direct antibacterial effects on intracellular and extracellular pathogens. Furthermore, mtROS contributes to the inflammatory response, whereas DRP1-mediated mitochondrial fission enhances tumor cell phagocytosis. Likewise, in M1 macrophages, L-arginine is metabolized by iNOS to produce nitric oxide, which has cytotoxic effects. On the other hand, M2 macrophages rely extensively on OXPHOS and FAO to perform their tissue-repairing and immunoregulatory functions. Unlike M1 macrophages, L-arginine metabolism in M2 macrophages is regulated by Arg-1, resulting in the generation of L-ornithine and polyamines, which stimulate tissue remodeling and repair. M1 and M2 macrophages'metabolic differences highlight the complex interplay between mitochondrial activity, metabolic pathways, and immune regulation. TCA; tricarboxylic acid, DRP-1; dynamin-related protein 1, iNOS; inducible nitric oxide synthase, OXPHOS; oxidative phosphorylation, FAO; fatty acid oxidation

Fig. 4

Involvement of mitochondria in the adaptive immune response: Mitochondrial ROS leads to increased production of inflammatory genes such as NF-ĸB, AP-1, and NFAT. In addition, mitochondria provide the essential components for protein synthesis. For example, MDH2 converts malate to oxaloacetate, which yields aspartate, a purine and pyrimidine precursor. Furthermore, SHMT2 offers one-carbon units for purine and thymidine synthesis, aiding T-cell proliferation. Excess lactate from the Warburg effect can convert to acetyl-CoA, which promotes IFN-γ production through histone acetylation. On the other hand, Treg differentiation is mostly dependent on OXPHOS rather than glycolysis; thus, inhibiting the glycolysis pathway lowers Th17 differentiation. During the memory phase, mitochondria join together to generate larger mitochondria. This remodeling relies on the fusion of proteins, including mitofusin-1, mitofusin-2, and OPA1, to increase OXPHOS and maintain SRC. The T cell exhaustion marker PD-1 signaling suppresses mitochondrial biogenesis by inhibiting the expression of PGC1α. Thus, PD-1 signaling inhibition may accelerate glucose absorption and improve mitochondrial metabolic performance. Regarding B cell proliferation, mitochondria activate swiprosin-2, an inner mitochondrial membrane protein that regulates metabolic switching during the transition from pro-B cells to pre-B cells. Memory B cells rely significantly on mitochondria for long-term survival. For instance, memory B cells are more resistant to mitochondrial apoptosis due to increased expression of the Bcl-2 anti-apoptotic proteins. Bcl-2 proteins prevent the release of apoptosis-inducing chemicals, such as cytochrome c and AIF, from the intermembrane gap. On the other hand, M2 macrophages rely extensively on OXPHOS and FAO to perform their tissue-repairing and immunoregulatory functions. Unlike M1 macrophages, L-arginine metabolism in M2 macrophages is regulated by Arg-1, resulting in the generation of L-ornithine and polyamines, which stimulate tissue remodeling and repair. M1 and M2 macrophages'metabolic differences highlight the complex interplay between mitochondrial activity, metabolic pathways, and immune regulation. AP-1; activating protein 1, NFAT; nuclear factor of activated T cells, MDH2; malate dehydrogenase-2, SHMT2; mitochondrial serine hydroxymethyltransferase 2, FOXP3; Forkhead box P3, ETC; electron transport chain, SRC; spare respiratory capacity, AIF; apoptosis-inducing factor, PD1; programmed cell death protein 1, PGC-1α, peroxisome proliferator-activated receptor-γ coactivator 1-α

Fig. 5

Mitochondria transferring mechanisms and tracking methods: A Transferring mechanisms; TNTs, the tube-like structures that connect cells, are formed with the crucial involvement of F-actin, myosin, and tubulin. These components play a significant role in transferring cellular components such as RNAs, proteins, and mitochondria. Mitochondria, for instance, move through TNTs by interacting with F-actin and the cytoskeleton. GJCs, on the other hand, are large protein channels linked to the cytoplasm of adjacent cells and are made of connexins. Six connexins form a connexon, and two connexons create a channel, exchanging small molecules and nutrients. Cx43, a specific connexin, helps mitochondrial transfer through Ca2 + or ROS exchange. EVs, like exosomes and MVs, also facilitate intercellular communication. Mitochondrial extrusion occurs when cells expel mitochondria freely or in vesicles, usually due to stress or damage. This mechanism depends on intact actin filaments and microtubules, which are crucial in maintaining mitochondrial quality control. B Mitochondria tracking methods: Fluorescent-based labeling uses mitochondrial dyes such as MitoTracker deep red and green to observe mitochondria in live cells. Transgenic reporter systems use GFP to track mitochondria with targeting sequences. Quantifying mtDNA with qPCR or sequencing tracks specific mitochondria. A new tool, MERCI, analyzes single-cell sequencing data to trace mtDNA. C Mitochondrial transfer and quality control using a robot-aided micro-manipulation system; The innovative technique to control the quality and quantity of mitochondria injected into single live cells using a robotic microneedle and optical tweezers system. This method arranges Mitochondria and cells in a 1-D array within a microfluidic device. The robotic microneedle, aided by optical tweezers, collects a set number of functional mitochondria and injects them into live cells without causing damage. TNT; Tunneling nano tubes, GJC; Gap junction channel Cx43; Connexin-43, ROS; Reactive oxygen species, EV; Extracellular vesicle, MV; Micro vesicle, mtDNA; Mitochondrial DNA, GFP; Green fluorescent protein, qPCR; Quantitative polymerase chain reaction

Fig. 6

Pro and anti-inflammatory effects of mitochondrial transfer. Transferring mitochondria has both pro- and anti-inflammatory effects. For instance, A transferring MSC mitochondria to CD4 + T cells can reduce Th1 proliferation and IFN-γ production by inhibiting T-bet. Also, CD8 + T cells that adopt MSCs'mitochondria exhibit lower growth, IFNγ production, and cytotoxic activities due to the downregulation of T-bet and Eomes transcription factors. This action is mostly performed by the IP3-AKT-mTOR pathway and glycolysis suppression. Furthermore, MSCs deliver mitochondria to T cells, enhancing the expression of genes associated with Treg development and activation, such as FOXP3, CTLA-4, CD39, and CD73. B Cancer cells share mutant mtDNA and fragmented mitochondria with T and NK cells in the tumor microenvironment, causing NK mitochondrial and cytotoxicity malfunction, T cell exhaustion, and senescence. Regarding the pro-inflammatory effects of Mito T, C it has been suggested that MT enhances the activation and longevity of elderly human CD4 + T cells, D transferring of mitochondria isolated from hepatocytes to NK cells, resulting in a significant increase in proliferation and elevated secretion of cytotoxic granules including granzyme B, perforin and IFN-γ, (E) it seems that under metabolically stressed conditions, adipose tissue MQs utilize exogenous mitochondria to support aerobic respiration and thermogenesis toward a pro-inflammatory phenotype, F platelets can transfer mitochondria to neutrophils via EVs to enhance activation, adhesion, and migration potential as well as elevating intracellular calcium and ROS levels in neutrophils, G the Mito T from MQs undergoing pyroptosis into neutrophils leads to increased mtROS production, lower MMP and activation of the Gasdermin D axis, ultimately triggering NETs formation. MT; mitochondrial transfer, MSC; mesenchymal stem cells, IP3; inositol 1,4,5-trisphosphate, mTOR; mammalian target of rapamycin

Fig. 7

Therapeutic effects of MT in pathological conditions. A Adaptive transfer of mito-transferred naive CD4 + T cells from old mice into Rag1-KO mice protected these mice against IAV and M. tb infections. B MSCs MT to Th17 reduce IL-17 production in RA, suggesting a novel mechanism for regulating Th17 cells in the inflammatory environment of RA. C MT enhances the function of elderly human T cells by increasing mitochondrial mass, modulating cytokine production, enhancing T cell activation, and reducing exhaustion markers to rejuvenate aged CD4 + T cells, potentially improving immune responses in elderly patients. D transfer of mitochondria from BM-SCs to CD8 + T cells expressing pmel-1 transgenic TCR to promote their antitumor activity against melanoma cells. E MSCs MT reduces early and late apoptosis following electroporation in CAR-T cells and shows an increased cytotoxic activity, potentially enhancing CAR-T treatment outcomes. F In mouse models, Mito + CD19-CAR T reduced leukemia cells and improved survival rates. MT; mitochondrial transfer, RA; rheumatoid arthritis, IAV; influenza A virus, M.tb; mycobacterium tuberculosis, BM-SCs; bone marrow stem cells