Diverse Roles of Mitochondria in Immune Responses: Novel Insights Into Immuno-Metabolism

Abstract

Lack of immune system cells or impairment in differentiation of immune cells is the basis for many chronic diseases. Metabolic changes could be the root cause for this immune cell impairment. These changes could be a result of altered transcription, cytokine production from surrounding cells, and changes in metabolic pathways. Immunity and mitochondria are interlinked with each other. An important feature of mitochondria is it can regulate activation, differentiation, and survival of immune cells. In addition, it can also release signals such as mitochondrial DNA (mtDNA) and mitochondrial ROS (mtROS) to regulate transcription of immune cells. From current literature, we found that mitochondria can regulate immunity in different ways. First, alterations in metabolic pathways (TCA cycle, oxidative phosphorylation, and FAO) and mitochondria induced transcriptional changes can lead to entirely different outcomes in immune cells. For example, M1 macrophages exhibit a broken TCA cycle and have a pro-inflammatory role. By contrast, M2 macrophages undergo β-oxidation to produce anti-inflammatory responses. In addition, amino acid metabolism, especially arginine, glutamine, serine, glycine, and tryptophan, is critical for T cell differentiation and macrophage polarization. Second, mitochondria can activate the inflammatory response. For instance, mitochondrial antiviral signaling and NLRP3 can be activated by mitochondria. Third, mitochondrial mass and mobility can be influenced by fission and fusion. Fission and fusion can influence immune functions. Finally, mitochondria are placed near the endoplasmic reticulum (ER) in immune cells. Therefore, mitochondria and ER junction signaling can also influence immune cell metabolism. Mitochondrial machinery such as metabolic pathways, amino acid metabolism, antioxidant systems, mitochondrial dynamics, mtDNA, mitophagy, and mtROS are crucial for immune functions. Here, we have demonstrated how mitochondria coordinate to alter immune responses and how changes in mitochondrial machinery contribute to alterations in immune responses. A better understanding of the molecular components of mitochondria is necessary. This can help in the development of safe and effective immune therapy or prevention of chronic diseases. In this review, we have presented an updated prospective of the mitochondrial machinery that drives various immune responses.

Keywords: TCA cycle; fatty acid oxidation; memory T cell; oxidative phosphorylation; regulatory T cell.

Figures

Figure 1

Mitochondria roles in immune responses: mitochondrial components are involved in immune functions. (1) Metabolic pathways such as TCA cycle, oxidative phosphorylation (OXPHOS), and fatty acid oxidation (FAO) are important for macrophage polarization and T cell differentiation. (2) Amino acid metabolism (Tryptophan metabolism is shown here.) also contributes to mitochondrial immune functions. Tryptophan depletion can cause immune tolerance. NAD+ is synthesized from tryptophan. NAD+ contributes to SIRT3-mediated mitochondrial biogenesis. In addition, NAD+ is required for metabolic pathways and CD4+ T cell differentiation. (3) Mitochondrial ROS (mtROS) control immune cell transcription, metabolism, and NLRP3 mediated inflammation. Antioxidants such as glutathione balance mtROS. SIRT3 is involved in regulating many mitochondrial proteins such as IDH2 (TCA cycle), MnSOD [reactive oxygen species (ROS) balance], and FOXO3. SIRT3 inhibits mtROS. (4) Mitophagy is crucial for removing damaged mitochondria. Damaged mitochondria are a source of mtROS so mitophagy balances mtROS. Pink1, localized on the mitochondrial outer membrane, binds to parkin and initiates mitophagy. Parkin mutations can increase the susceptibility to the intracellular bacteria Mycobacterium leprae and Salmonella enterica. Decreased mitophagy results in increased ROS which further increases the susceptibility to infections. Hepatitis B and C viruses use mitophagy to their benefit. These viruses protect themselves from mitochondria induced apoptosis by activating mitophagy. (5) Mitochondrial antiviral signaling (MAVS) in the mitochondria membrane initiates the inflammatory response. Upon viral infections, viral RNA is sensed by RIG-I. RIG-I activates MAVS (located on the outer membrane of mitochondria). MAVS signaling can activate NLRP3 inflammasome and NF-κB/IRF transcription. (6) NLRP3 inflammasome contributes to the activation of caspase-1 and leads to NF-κB signaling and production of IL-1β and IL-18. (7) Mitochondrial DNA (mtDNA) can be released from mitochondria into the cytosol and activate the NLRP3 inflammasome and production of IL-1β and IL-18. Also, mtROS can induce mtDNA mutation. (8) Mitochondrial dynamics, fission (OPA-1) and fusion (Drp1), is associated with activated T cells and memory T cells, respectively. (9) ER and mitochondria junction: in CD8+ memory T cells, mitochondria are placed near the ER. HK-1 (hexokinase-1, an important enzyme in glycolysis) in conjunction with pAKT, mammalian target of rapamycin complex 2 (mTORC2), and glycogen synthase kinase 3β (GSK-3β) mediated signaling cascades regulate CD8+ memory T cell metabolism. (10) Uncoupling protein 2 (UCP2), a mitochondrial membrane protein, allows protons to enter the mitochondrial matrix, thereby decreasing mitochondrial membrane potential which further decreases mtROS.

Figure 2

Metabolic regulation in macrophages and T cells: (A) metabolism during macrophage polarization: polarization of monocytes to M1 or M2 phenotypes is controlled by the cytokines produced by other immune cells. M1 macrophages are activated by IFN-γ produced by Th1 cells. M1 macrophages tend to store surplus FA as triacyclglycerols and cholesteryl esters in lipid droplets, and they exhibit higher aerobic glycolysis and lower oxidative phosphorylation (OXPHOS). Nitric oxide production is higher in M1. Uncoupling protein 2 (UCP2) expression is decreased in M1 macrophages. Contrarily, M2 macrophages are activated by IL-4 or IL-13 to regulate anti-inflammation and promote Th2 response and tissue repair. M2 macrophages adopt a metabolic program dominated by fatty acid-fueled OXPHOS and channel FA toward re-esterification and β-oxidation. Silencing UCP2 impairs M2 macrophage activation by IL-4. High adenosine monophosphate-activated protein kinase (AMPK) and low NO is the reason for high OXPHOS in M2 macrophages. (B) Metabolism during T cells differentiation: naïve T cells are dependent on OXPHOS as their primary metabolic pathway. By contrast, activated T cells exhibit higher glycolysis than OXPHOS. After differentiation, Th1, Th2, and Th17 have higher glycolysis than OXPHOS and high mTORC1 activity. Memory T cells and regulatory T cells undergo AMPK-dependent FAO and have variable mTORC1.

Figure 3

Metabolism in M1 macrophages. (A) M1 macrophage metabolic regulation: M1 macrophages are prevalent in obese adipose tissue. Glucose uptake is increased in M1 macrophages. Importantly, the TCA cycle exhibits two breaks. The first break involves the enzyme isocitrate dehydrogenase (IDH) which results in increased levels of citrate and itaconic acid. Citrate feeds fatty acid (FA) synthesis for prostaglandin (PG) and nitric oxide (NO) production while itaconic acid has anti-bacterial properties. The second break happens with the enzyme succinate dehydrogenase (SDH) which causes increased succinate levels. Succinate stabilizes HIF-1α which binds to the interleukin (IL)-1β promoter boosting IL-1β production and inflammation. Increased flow through the aspartate arginosuccinate shunt (AASS) replenishes the TCA cycle which further increases citrate levels and feeds the urea cycle which contributes to NO production. Glutamine is converted into glutamate by glutamate synthase (GS). Glutamate can further be converted into α-ketoglutarate (αKG). Low αKG/succinate ratio strengthens M1 macrophage activation. Glutamine-synthetase inhibition skews M2-polarized macrophages toward the M1-like phenotype characterized by reduced intracellular glutamine and increased succinate with enhanced glucose flux through glycolysis which is partly related to HIF-1α activation. (B) M1 macrophage transcriptional regulation: in M1 macrophages, the TCA cycle is broken. As a result, citrate is converted to free fatty acids (FFAs). FFAs activate macrophage IKK and JNK1 signaling molecules to induce M1 polarization. Activation of IKK and JNK1 in macrophages produces IL-1β and TNFα which can initiate inflammatory response. Adiponectin is released from adipose tissue and is involved in the breakdown of fatty acids. Adiponectin signaling inhibits M1 programming.

Figure 4

Metabolism in M2 macrophages. (A) M2 macrophage metabolic regulation: M2 macrophages adopt a metabolic program fueled by fatty acids and oxidative phosphorylation (OXPHOS). M2 macrophages are seen in lean adipose tissue and lipid uptake is increased in M2 macrophages (8). Both OXPHOS and FAO are important for the anti-inflammatory function of M2 macrophages. In M2 macrophages, glycolysis is impaired due to low HIF-1α activity and low expression of the active 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (PFKFB1). Also, high α-KG/succinate ratio promotes the M2 phenotype. This could be due to the high flow of the TCA cycle or glutamine metabolism. M2 macrophages will channel FA toward re-esterification and β-oxidation. β-oxidation is channeled through carinitine palmitoyl transferase 1 (CPT1) (9). The high rates of OXPHOS in M2 macrophages are caused by low nitric oxide (NO) production and high adenosine monophosphate-activated protein kinase (AMPK) activity. (B) M2 macrophage transcriptional regulation: omega-3 polyunsaturated fatty acids (PUFA) undergo enzymatic transformation by 12/15-LO (lipoxygenase) and other enzymes in the production of specialized pro-resolving lipid mediators (SPMs) and intermediate metabolites that are potent PPAR ligands. M2-inducing cytokines IL-4 and IL-13 promote STAT6 phosphorylation to induce transcription of PPARs (γ and δ) and their coactivator PPARγ-coactivator-1β (PGC-1β). M2 exhibit enhanced PPAR-γ activity. PPAR-γ forms a heterodimer with RXR to induce OXHOS regulating proteins. PGC-1β promotes the STAT6 transcription complex and expression of M2 proteins such as arginase-1 and the pattern-recognition/endocytic receptor CD206. The STAT6 transcription complex further increases transcription of TCA cycle enzymes such as citrate synthase, CD36 (fatty acid transporter), ATP synthase, 12/15 lipoxygenase (converts omega-3 PUFA to SPMs), and LCAD (long chain acyl dehydrogenase, an enzyme for β-oxidation). Adiponectin activates PPARs in M2 macrophages and consequently increases β-oxidation.

Figure 5

Tryptophan metabolism mediated immune cell response. L-tryptophan (Trp) can only be obtained in the human body from dietary sources. It is catabolized by the enzyme indoleamine-2,3-dioxygenase-1 (IDO-1) to L-kynurenine (Kyn). Interferon-γ produced by Th1 cells stimulates IDO-1 production in antigen-presenting cells (APCs). Tumor cells are also known to have increased production of IDO-1. Increases in IDO-1 cause conversion of Trp to Kyn resulting in lower Trp and higher Kyn in plasma. Kyn is a ligand for aryl hydrocarbon receptor (AhR). Mitochondrial AhR (MitoAhR) is localized in the inner membrane space of mitochondria and is also affected by Kyn. (1) Immune tolerance by kynurenine: increased Kyn causes immune tolerance by inhibiting proliferation of both T cells and NK cells by inhibiting maturation of dendritic cells and increasing proliferation of immune suppresser cells (Treg and MDSC). Tryptophan depletion also causes apoptosis of T cells due to nutrient deficiency. (2) NAD+ mediated immune function: in addition, Trp is one of the sources for NAD+ biosynthesis. NAD+ is an important metabolite for metabolic processes [glycolysis, TCA cycle, and oxidative phosphorylation (OXPHOS)] and production of NADPH. During oxidative burst in macrophages, NAPPH is oxidized in the presence of NADPH oxidase to produce superoxide which is utilized to kill pathogens. NAD+ contributes to CD4+ T cell differentiation and SIRT3-mediated mitochondrial biogenesis. (3) mTOR inhibitor rapamycin can be used to treat systemic lupus erythematosus (SLE) and mitochondrial disease: T cell dysfunction is associated with SLE. In SLE, an increase in kynurenine and kynurenine mediated mTOR stimulation was observed. In addition, accumulation of reactive oxygen species (ROS) producing mitochondria and decreased Drp1 are associated with SLE. Rapamycin can increase Drp1 by inhibiting mTORC1. (4) Rapamycin inhibits mitochondrial ROS (mtROS): rapamycin inhibits mtROS through several mechanisms. Complex I in mitochondria is a source of ROS generation. Rapamycin can inhibit mtROS production by inhibiting complex I formation. In addition to this, rapamycin can inhibit mtROS by inducing autophagy and activating p62 and the NRF2 pathway.

Figure 6

Mitochondria related signal cascades to activate immunity. Mitochondrial antiviral signaling (MAVS) is activated by the viral RNA sensor RIG-I and promotes inflammatory and immune gene expression via transcription factor NF-κB and IRFs. MAVS receptors can be found in the mitochondrial outer membrane. Mitochondrial ROS (mtROS) are also able to activate MAVS independently of RNA. The NLRP3 inflammasome also signals from the outer mitochondrial membrane. Like MAVS, NLRP3 also responds to mtROS and can cause mitochondrial damage that promotes the generation of additional ROS. NLRP3 drives the production of IL-1β and IL-18. Mitochondrial DNA can also activate NLRP3. Finally, MAVS promotes oligomerization of NLRP3 at the mitochondria. Therefore, two major innate immune pathways (RIG-I/MAVS and NLRP3) are all dependent on mitochondria.

Figure 7

Mitochondrial fission and fusion contribute to immune responses. Mitochondrial fission and fusion controls mitochondrial mass. Nutrient deprivation induces an increase in mitochondrial fusion and suppression of mitophagy. On the other hand, prolonged DNA damage leads to mitochondrial fission. Fusion (tubular mitochondria) is controlled by two dynamin-like GTPases for the outer membrane proteins (mitofusin MFN1 and MFN2) and inner membrane proteins (OPA1). Fission (fragmented mitochondria) is coordinated by four outer membrane proteins (Drp1, Mff, Mid49, and mid52). Fusion and fission of mitochondria contribute to immune response. (1) Memory T cells have increased fusion generating more oxidative phosphorylation (OXPHOS) and FAO. (2) Clearance of apoptotic cells by phagocytes is known as efferocytosis. Resting macrophages have displayed tubular mitochondria. (3) Fission contributes to mitochondria mobility. During antigen-specific T cell and NK cell activation, mitochondria fission increased, and mitochondria move toward the immune synapse (IS) and facilitate ATP production at the IS. (4) Uptake of apoptotic cells by macrophages during efferocytosis requires fission and increased Drp1. (5) In mouse-derived bone marrow macrophages, knocking down Drp1 initiates NLRP3 inflammasomal activation, and inducing mitochondrial fission attenuated NLRP3 inflammasomal assembly and activation. (6) Activated effector T cells have increased mitochondrial fission and exhibit higher rates of glycolysis.

Figure 8

Reactive oxygen species (ROS)-mediated cell signaling in T cells: low/moderate levels of ROS promote mTOR, Myc, and nuclear factor of activated T cell (NFAT) activation which is associated with increased glycolysis and inflammatory response in T cells. ROS levels are regulated by the glutathione pathway (GSH) and NRF2–Keap1–Cul3 trimeric complex. Abrogation of GSH and the trimeric complex, which may cause high levels of ROS, impairs the inflammatory response of T cells. In addition, high ROS and high adenosine monophosphate-activated protein kinase (AMPK) levels inhibit mTOR. It is reported that naïve T cells, CD4+ regulatory T cells, and chronically stimulated T cells depend on OXPHOS as their primary metabolism method while CD8+ effector and CD4+ Th1 cells mainly depend on glycolysis. This metabolic switching is controlled by ROS.

Figure 9

Antioxidants neutralize mitochondrial ROS (mtROS). (1) Antioxidants in mitochondria: superoxide (O2−) is released from complex I (into the matrix) and III (into both the matrix and intermembrane space). This is converted to H2O2 in the presence of superoxide dismutase (SOD). Mn-SOD (manganese dismutase) is localized in mitochondria matrix and Cu/Zn-SOD (in the intermembrane space and cytosol). SIRT3 is involved in deacetylation of MnSOD. Catalase converts H2O2 to H2O. Catalase is also localized in mitochondria (MCAT). Glutathione converts OH− to H2O to reduce the cellular damage. Glutathione (GSH) is oxidized and reduced to glutathione disulfide (GSSG) in a reversible reaction. NADPH from the pentose phosphate pathway reduces GSSG to form GSH in presence of glutathione peroxidase (Gpx). (2) Antioxidants play an important role in neutralizing mtROS. Uncoupling protein 2 (UCP2) (mitochondrial uncoupler protein), which helps to shuttle H+ ions from the intermembrane space to the mitochondria matrix, decreases the membrane potential to inhibit excessive reactive oxygen species (ROS) production. Excessive ROS production can impact ATP synthesis, cytokine production, mitochondrial DNA (mtDNA) mutation, and post-translational modification of cellular proteins by S-nitosylation or glutathionylation.