Mitochondria: A master regulator in macrophage and T cell immunity

Accepted Manuscript Mitochondria: A master regulator in macrophage and T cell immunity

Pu-Ste Liu, Ping-Chih Ho PII: DOI: Reference:

S1567-7249(17)30214-3 doi:10.1016/j.mito.2017.11.002 MITOCH 1239

To appear in:

Mitochondrion

Received date: Revised date: Accepted date:

13 August 2017 30 October 2017 3 November 2017

Please cite this article as: Pu-Ste Liu, Ping-Chih Ho , Mitochondria: A master regulator in macrophage and T cell immunity. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mitoch(2017), doi:10.1016/ j.mito.2017.11.002

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ACCEPTED MANUSCRIPT Mitochondria: a master regulator in Macrophage and T cell immunity Pu-Ste Liu1,2 and Ping-Chih Ho1, 2 1

Department of Fundamental Oncology, Faculty of Biology and Medicine, University of

Lausanne, Epalinges, Vaud, Switzerland

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Ludwig Lausanne Branch, Epalinges, Vaud, Switzerland

* Correspondence author at:

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2

Department of Fundamental Oncology, University of Lausanne

Ludwig Lausanne Branch, 155 Ch. des Boveresses, 1066 Epalinges, Switzerland. Email address: [email protected] (P.-S. L.)

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[email protected] (P.-C. H.)

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1. Abstract Orchestrating biological activities of immune cells through metabolic reprogramming reveals a new approach to harnessing immune responses. Increasing evidence reveals that the mitochondrion is a central regulator for modulating metabolic reprogramming and controlling immune cell activation and functions. In addition to supporting bioenergetic demands, the mitochondrion serves as a signaling platform through the generation of reactive oxygen

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species and metabolites of the tricarboxylic acid cycle to modulate signaling cascades controlling immune cell activation and immune responses. Herein, we discuss the mechanisms through which the mitochondrion acts as a master regulator to fine-tune immune responses elicited by macrophages and T cells. Keywords

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Mitochondria, cellular metabolism, immunity, Macrophages, T cells, PRRs

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2. Introduction The mitochondrion, the cellular powerhouse of eukaryotes, generates adenosine triphosphate (ATP) through the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), in which this organelle oxidizes nutrients such as pyruvate, glutamine, and fatty acids (FAs). In addition to ATP production, the mitochondrion is an important source of metabolic

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intermediates required for macromolecular synthesis (protein and FA synthesis) and for posttranslational modification of signaling proteins (phosphorylation and acetylation). Moreover, mitochondrial ETC complexes are major sites of reactive oxygen species (ROS) production and for controlling the NAD/NADH ratio, two critical events that orchestrate cellular processes (Wai and Langer, 2016). Given that the mitochondrion controls multiple cellular

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processes, it was suggested to serve as a signaling orga nelle to bridge cellular metabolism with cellular processes in response to nutrient availability and cellular metabolic demands

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(Chandel, 2014).

Recently, mounting evidence reveals that the mitochondrion has a crucial role in innate

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and adaptive immunity. In addition to supporting energy production, cellular metabolism was revealed to fine-tune immune cell activation and function (O'Neill et al., 2016). For example, naive and alternatively activated (M2) macrophages predominantly use oxidative

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phosphorylation (OXPHOS), whereas classically activated (M1) macrophages robustly increase the activity of aerobic glycolysis and the pentose phosphate pathway (O’Neill and

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Pearce, 2016). In T cells, activation induces a metabolic switch that in turn participates in proliferation and differentiation (Buck et al., 2015). In these processes, mitochondria act as a

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central regulator, and emerging evidence suggests that mitochondria-derived regulations can be critical for immunometabolic regulations in immune cells (Chao et al., 2017; Mehta et al., 2017). In this review, we examine the underlying mechanisms through which mitochondria regulate immune responses in both macrophages and T cells. Furthermore, we discuss how mitochondria-derived regulations can be harnessed to control immune responses under pathological conditions.

3. Macrophage immunity Macrophages are essential components of innate immunity and represent the first line of defense against pathogens. Moreover, macrophages can orchestrate tissue homeostasis upon

ACCEPTED MANUSCRIPT tissue damage with anti- inflammatory responses. In this section, we show that mitochondria not only function as signaling platforms to participate in pattern recognition receptor (PRR)induced signaling but also provide metabolites that regulate macrop hage activation and functions.

3.1 Mitochondrion: a signaling platform for macrophage activation and functions PRRs, controlling classic activation of macrophages, consist of four protein families: Toll-

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like receptors (TLRs), nucleotide oligomerizatio n domain- like receptors (NLRs), C-type lectin receptors, and retinoic acid- inducible gene I (RIG-I)-like receptors (RLRs) (Kieser and Kagan, 2017). These receptors detect pathogen-associated molecular patterns, such as microbial structural components, nucleic acids and proteins, or damage-associated molecular patterns (DAMPs) released from injured cells. Upon ligation with ligands of PRRs, specific

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cellular signaling cascades are activated for producing inflammatory cytokines (Akira et al., 2006). In addition to these canonical signaling cascades, metabolic switch, including aerobic glycolysis and alterations in OXPHOS, crucially fine-tunes the strength and duration of

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macrophage immune responses. Furthermore, several downstream effectors and adaptors of PRRs have been suggested to function in proximity with the mitochondrial outer membrane

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(Arnoult et al., 2011). For example, following the stimulation of TLRs (TLR1, TLR2, and TLR4), TRAF6 interacts with ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), a protein that has been implicated in mitochondrial respiratory complex I

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assembly, to promote mitochondrial ROS (mtROS) production for antibacterial responses (Fig. 1a).(West et al., 2011) Moreover, upon mitochondrial damage, mtDNA is released into

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the cytoplasm, which in turn leads to activation of the NFκ-B signaling pathway and the induction of multiple proinflammatory genes including TNFα and IL-6 (Fig. 1a) (Zhang et al.,

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2014). In addition to TLRs, RLRs support viral immunity by engaging a mitochondrial antiviral signaling (MAVS)- induced antiviral response (Fig. 1b) (Pourcelot and Arnoult, 2014; Seth et al., 2005; Vazquez and Horner, 2015). Mechanistically, MAVS localized on the outer mitochondrial membrane (OMM) acts as a critical adaptor protein and coordinates signals received from two independent cytosolic RLRs (RIG-I and MDA5) to induce the production of type I IFNs and proinflammatory cytokines (Vazquez and Horner, 2015). Once receiving activation signals, MAVS recruits TRAF5, TRAF6, and TRADD to activate the IκB kinase (IKK) complex that in turn stimulates NF-κB- mediated transcription of proinflammatory cytokines (Tang and Wang, 2010). Furthermore, MAVS interacts with TRAF2/3 and TANK to promote IKKε and TBK1 activation for inducing type I interferon

ACCEPTED MANUSCRIPT production (Michallet et al., 2008). Another mitochondrial protein, TOM70, also interacts with MAVS to regulate the IKKε-TBK-IRF3/7 pathway (Liu et al., 2010). Notably, STING (stimulator of interferon genes), a protein localized on endoplasmic reticulum (ER)– mitochondrial contact sites, can coordinate with MAVS to stimulate signaling cascades required for RLR signaling and antiviral responses (Ishikawa and Barber, 2009). Even though these molecular mechanisms governing immune responses have been revealed, it remains elusive as to how these interactions between different RLR signaling pathways are integrated

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in mitochondria in response to specific types of viral infection and whether these regulations can be controlled by metabolic insults in mitochondria.

The NLRP3 inflammasome is complex, consisting of NLRP3, the adaptor protein ASC (apoptosis-associated speck-like protein), and the effector protein cysteine protease caspase-1 (Zhou et al., 2011). NLRP3 inflammasome assembly activates caspase 1 to stimulate the

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maturation process of pro-IL-1β for secretion. In addition to DAMPs, mtROS production and damaged mtDNA have been shown to induce NLRP3 activation and subsequent NLRP3 inflammasome assembly (Zhou et al., 2011). Notably, the accumulation of damaged

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mitochondria potentiates NLRP3-dependent inflammasome activation, but NF-kB-induced mitophagy can restrain NLRP3- induced inflammatory responses in macrophages (Zhong et

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al., 2016). MAVS-mediated recruitment of the NLRP3 inflammasome to mitochondria is critical for IL-1β production and NLRP3 and pro-IL-1β expression for sustained inflammatory responses (Subramanian et al., 2013). Furthermore, several OMM associated

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proteins have been shown to affect NLRP3 inflammasome activation. For example, mito fusin 2, a mitochondrial outer membrane protein required for mitochondrial fusion, activates the

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NLRP3 inflammasome during influenza and encephalomyocarditis virus infection (Ichinohe et al., 2013). Hexokinase is not only a key glycolytic enzyme but also associated with the

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VDAC (voltage dependent anion channel) in OMM for regulation of glycolysis, mitochondrial

stability,

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production,

and

permeability

transition

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formation(Pastorino and Hoek, 2008). Inhibition of the glycolytic hexokinase by Nacetylglucosamine (NAG), the degradation of S. aureus cell wall peptidoglycan (PGN) results in hexokinase dissociation from the OMM also triggers NLRP3 activation and subsequent inflammatory IL-1β secretion (Wolf et al., 2016). In addition, the mitochondrial lipid cardiolipin could be recruited to the mitochondrial outer membrane, which is required for NLRP3 activation in response to ROS-dependent and ROS- independent activation.(Iyer et al., 2013) Despite these findings, the integrated protein complex on the OMM to distinguish

ACCEPTED MANUSCRIPT stimuli and determine the activation duration of the NLRP3 inflammasome remains largely unknown (Gurung et al., 2015).

3.2 Mitochondrial metabolism instructs macrophage activation and inflammatory responses. Macrophages stimulated with lipopolysaccharides (LPS), in the presence or absence of interferon gamma (IFN-r), differentiate into M1 macrophages characterized by elevated

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production of proinflammatory molecules such as IL1β, TNFα, IL-6, IL-12, and ROS. By contrast, IL-4 or IL-13-stimulated macrophages become M2 macrophages exhibiting antiinflammatory activity for maintaining tissue homeostasis and repair and against helminth infections. In addition to TLR- and cytokine-mediated signaling cascades on orchestrating macrophage activation, emerging results have revealed that cellular metabolisms fine-tune

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macrophage phenotypes and functions through bioenergetic regulations ( Fig. 2) (O’Neill and Pearce, 2016). M1 macrophages increase glucose uptake and exhibit enhanced aerobic glycolysis, whereas FA uptake and OXPHOS are enhanced to support M2 macrophage

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polarization and functions. The mitochondrion is a central metabolic organelle that governs the dependence of cellular metabolism and the coordination of the TCA cycle and ETC to

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modulate OXPHOS rates and ATP production, as well as the generation of important intermediates to support other metabolic processes. In addition to increased glycolysis, M1 macrophages have an impaired TCA cycle with two broken steps. The first deficient step is at

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isocitrate dehydrogenase 1 (IDH1), which converts isocitrate to α-ketoglutarate (αKG). The deficiency of IDH1 in M1 macrophages results in the accumulation of citrate, which can

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further be exported to cytosol for FA biosynthesis and nitric oxide production. Citrate can also be used for itaconate production through immune-responsive gene 1 (IRG1). Notably,

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itaconate inhibits succinate dehydrogenase (SDH), thereby inducing a second break that causes succinate to accumulate (Hall et al., 2013; Lampropoulou et al., 2016). SDH is not only the TCA enzyme but also functions as an electron carrier for complex II of the ETC, which is associated with mtROS production. Additionally, succinate, an antagonist for PHD, stabilizes hypoxia- inducible factor 1 α (HIF1α), inducing IL-1β expression(Tannahill et al., 2013). Moreover, increased SDH activity and mitochondrial hyperpolarization also induce increased mtROS production contributing IL1β expression by subsequently activates HIF-1α in LPS activated macrophages (Mills et al., 2016). In addition to metabolite-derived regulation, modulation of ETC complex assembly plays a critical role in orchestrating macrophage activity. The ETC consists of two electron carriers and four respiratory

ACCEPTED MANUSCRIPT complexes (CI–IV), and these complexes (except CII) can assemble into supercomplexes that robustly enhance the ETC rate. Treating macrophages with bacterial products or living bacteria enhances ROS production by suppressing supercomplex formation (Garaude et al., 2016). Moreover, studies have indicated that LPS- induced NO production impairs ETC function and reduces OXPHOS activity, a critical regulation to prevent phenotype switch in M1 macrophages (Mills et al., 2016; Van den Bossche et al., 2016).

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In contrast to classical activation of macrophages, M2 macrophages have an intact TCA

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cycle and involve IL-4-mediated induction of FA oxidation (FAO) and mitochondrial biogenesis, thus sustaining anti- inflammatory responses through affecting the STAT6 and PGC-1β signaling pathways (Vats et al., 2006). Moreover, CD36- mediated uptake of triglycerides and their subsequent liposomal lipolysis support OXPHOS and the expression of M2 marker genes in IL-4-stimulated macrophages (Huang et al., 2014). However, recent

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works uncovered that glycolysis also has a crucial role in M2 macrophages and glucose oxidation supports FA synthesis for increasing FAO in M2 macrophages (Huang et al., 2016).

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IL-4 induced ATP citrate lyase activation and OXPHOS also enhance acetyl-CoA synthesis, contributing to histone acetylation, a critical epigenetic regulation for certain M2 marker gene expression (Covarrubias et al., 2016). In addition to FA and glucose metabolism, glutamine

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metabolism is essential for M2 macrophage activation through UDP-GlcNAC production(Jha et al., 2015). Notably, our recent studies have revealed that glutaminolysis fine-tunes M1 and

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M2 activation through the balance of succinate and α-ketoglutarate production. Mechanistically, α-ketoglutarate boosts FAO-dependent OXPHOS and the epigenetic

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reprograming of the M2 gene through Jumonji domain containing 3 (Jmjd3)-dependent regulations (Liu et al., 2017). How mitochondria respond to M2 stimulation and the

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mechanisms through which mitochondria-derived regulations modulate M2 macrophage immune responses remain largely unknown. Altogether, macrophages function in antipathogen host defenses, in addition to being leading cells affecting inflammatory disease progression such as obesity, atherosclerosis, and cancer (Geeraerts et al., 2017). Additionally, macrophages are considered dynamic cells that adapt their phenotype, and also possibly their metabolic state, through different disease phases. Because macrophages display different demands when they differentiate into pro- and anti- inflammatory macrophages, manipulating metabolic circuits has been suggested as a new therapeutic approach for educating macrophages in different disease contexts (Biswas and Mantovani, 2012; O’Neill and Pearce, 2016).

ACCEPTED MANUSCRIPT 4. T cell immunity A number of reviews have detailed the various changes in T cell metabolism after T cell activation, expansion, contraction, and differentiation.(Pearce et al., 2013) In this section, we will focus on studies integrating T cell metabolism into mitochondria and how mitochondrial metabolism supports T cell activation, proliferation, and differentiation as well as

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determination of their fate and function.

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4.1 Mitochondrial metabolism plays an important role in T cell activation. Quiescent naive T cells have a lower rate of nutrient uptake and rely on OXPHOS to generate ATP to support their survival. Upon antigen recognition, activated T cells increase glycolysis to support their proliferation and differentiation. Despite adopting an increased glycolytic metabolism, augmented mitochondrial metabolism also plays a crucial role in supporting T

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cell activation and proliferation (Fig. 3) (Mehta et al., 2017; Mills et al., 2017). For example, pyruvate dehydrogenase promotes pyruvate->acetyl-CoA conversion to support IL-2

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production and T cell activation and proliferation (Gerriets et al., 2015). Moreover, T cell activation induces lactate dehydrogenase (LDHA) expression, which promotes cytosolic levels of acetyl-CoA by shunting pyruvate into citrate through the TCA cycle, and the

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production of acetyl-CoA determines Th1 differentiation through epigenetic reprogramming (Peng et al., 2016). However, it remains unclear how LDHA directly modulates acetyl-CoA

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levels, whether by increasing levels of citrate available for conversion to acetyl-CoA or through promoting acetyl-CoA synthetase. Moreover, increased acetyl-CoA could be used for

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boosting histone acetylation and transcription of IFNr in CD8 + effector memory T cells.(Gubser et al., 2013) Mitochondria also modulate T cell activation and proliferation

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through glutaminolysis, in which αKG production is believed to be a critical step, but the detailed mechanisms remain elusive (Carr et al., 2010; Wang et al., 2011). Furthermore, upon T cell activation, various mitochondrial proteins are induced to boost biogenesis of specialized mitochondria, which supports folate-dependent one-carbon metabolism (RonHarel et al., 2016). In contrast to supporting T cell activation and differentiation, mitochondrial metabolism in T cells has been shown to influence aberrant T cell activity in autoimmunity and drive T cell exhaustion. For example, alloreactive T cells increase FA uptake and FAO in the graft- versus- host disease (GVHD) model, and targeting FAO in alloreactive T cells could ameliorate GVHD progression.(Byersdorfer et al., 2013) In addition, ligation of programmed cell death protein 1 (PD-1) in activated T cells shifts

ACCEPTED MANUSCRIPT metabolic preference from glycolysis to FAO and OXPHOS, and this metabolic shift could lead to T cell exhaustion through undefined molecular mechanisms. (Bengsch et al., 2016) Mitochondria could also impact T cell activation, proliferation, and effector function through ROS production.(Murphy and Siegel, 2013) In particular, ROS generated by complex III of the ETC function as a secondary messenger to modulate the Ca 2+-NFAT signaling cascade, which is critical for T cell activation (Quintana et al., 2007). Moreover,

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mitochondria were shown to accumulate at the immunological synapse (IS) during T cell

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activation and then sustain the Ca2+-NFAT signaling cascade through mitochondria-mediated Ca2+ buffering and enhance mtROS production (Quintana et al., 2007). Consistent with this finding, the mitochondrial enzyme glycerol-3-phosphate dehydrogenase is activated by Ca 2+ and was proposed as the source of mtROS during T cell activation.(Kamiński et al., 2012) In addition to supporting T cell activation, increased mitochondrial OXPHOS is critical to

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prevent activation- induced cell death. As demonstrated in a recent study, CD4 + T cells’ lack of cytochrome C oxidase (COX), an assembly molecule for complex IV of ETC, maintains

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their ability to engage aerobic glycolysis upon T cell activation; however, the unmatched OXPHOS, due to COX deficiency, promotes activation- induced cell death (Tarasenko et al.,

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2017).

4.2 Mitochondrial metabolis m and dynamics regulate memory CD8 + and regulatory T cell formation

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When infection is cleared, effector CD8 + T cells undergo a contraction phase, and only a small fraction of population survives to become long- lived memory CD8+ T cells. Memory

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CD8+ T cells prefer to utilize FAO to sustain bioenergetic demands (van der Windt et al., 2012). Moreover, memory T cells increase mitochondrial mass and spare respiratory capacity

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to facilitate OXPHOS; this metabolic feature is believed to enable memory T cells to respond more rapidly upon secondary exposure to the antigen (van der Windt et al., 2012). Memory CD8+ T cells also upregulate carnitine palmitoyltransferase 1 α to boost FA transport across the OMM, a rate- limiting step in FAO (van der Windt et al., 2012). Notably, memory T cells engage de novo lipogenesis to produce lipids from glucose, rather than obtain lipids directly from an extracellular environment (Cui et al., 2015; O’Sullivan et al., 2014). This unique metabolic process is energy- inefficient, and whether this futile cycle would provide unexplored metabolic checkpoints to ensure proper memory T cell differentiation remains unclear.

ACCEPTED MANUSCRIPT In addition to mitochondrial metabolism- mediated regulations on memory T cell differentiation, mitochondrial dynamics has recently been revealed to provide a new layer of regulation (Buck et al., 2016). Mitochondrial dynamics refers to the changes in mitochondrial size, shape, and cellular localization. Mitochondrial fusion and fission are two major steps that orchestrate proper mitochondrial size and shape in response to metabolic demands. Fission is regulated by Drp1 and Fis1, and fusion is controlled by OpaI, Mfn1, and Mfn2 (Chao et al., 2017). In contrast to effector T cells displaying fragmented mitochondria with

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looser cristae, memory T cells have elongated mitochondria with densely packed cristae (Buck et al., 2016). These changes in mitochondrial structure remodel mitochondrial properties and ETC complexes to meet metabolic demands in effector and me mory T cells. Mechanistically, OPA1 supports formation of tight cristal organization and facilitates ETC complex assembly for boosting ETC and OXPHOS activity. In the other worlds, the loss of

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Opa1, but not Mfn1 or Mfn2, decreased survival of memory T cells, which was associated with altered cristae structure and decreased spare respiratory capacity. Importantly, effector T cells could be shifted to becoming memory T cells and accordingly change their nutrient

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preference to FAO by elongating mitochondria either by Opa1 overexpression or the combined treatment with two compounds, one that blocked fission (Mdivi1) and one

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promoting fusion (M2). In contrast to fusion, the mitochondrial fission factor Drp1 modulates mtROS generation and controls mitochondrial localization at the IS during T cell activation, which in turn regulates IS formation and downstream TCR signaling (Röth et al., 2014).

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Furthermore, genetic ablation of the methylation-controlled J gene (MCJ), an endogenous inhibitor of mitochondrial complex I, in T cells promotes ETC supercomplex assembly,

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OXPHOS, and IFNr and IL-2 secretion (Champagne et al., 2016). Together, these findings suggest that mitochondrial dynamics controls CD8 + T cell differentiation and effector

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function; however, how these processes can be utilized for vaccine design and whether pathogens can impair CD8+ T cell- mediated immune response by hijacking these processes remain unclear.

Activated CD4+ helper T cells differentiate into Th1, Th2, Th17, and Treg cells in response to cytokine milieus. Emerging evidence suggests that differentiation of effector populations is intertwined with metabolic reprogramming (Almeida et al., 2016; Windt and Pearce, 2012). Aerobic glycolysis supports differentiation of Th1, Th2, and Th17 cells (Frauwirth et al., 2002; Michalek et al., 2011; Wang et al., 2011). Conversely, Treg cells engage

FAO

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expression

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sustain

their

ACCEPTED MANUSCRIPT immunosuppressive ability. Moreover, Foxp3 suppresses Myc signaling and glycolysis, but it enhances OXPHOS and NAD+ regeneration,(Angelin et al., 2017) suggesting that the balance between mitochondrial metabolism and aerobic glycolysis is a critical event to ensure Treg lineage stability. However, whether mitochondrial activity and dynamics in Tregs can be altered in response to the inflammatory status in the peripheral tissue remains unknown.

5. Conclusion and Future directions

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Evidence suggests that cellular metabolism is an important determinant of the functional phenotype of immune cells under a physiological and pathological environment(Bengsch et al., 2016; Geeraerts et al., 2017). The mitochondrion acts as a central immune regulator to integrate metabolic demands associated with signaling cascades and gene expression of immune cells. However, the detailed mechanisms through which mitochondria integrate these

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cellular processes to fine-tune immune response in both innate and adaptive immune cells must be identified. A comprehensive understanding of how mitochondria dictate specific immune cells’ activation, differentiation, and function may facilitate the discovery of novel

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therapeutic strategies for the treatment of human disease.

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Acknowledgements

This study was supported in part by SNSF project grant (31003A_163204), ISREC grant

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(26075483), Swiss Cancer Foundation (KFS-3949-08-2016), Harry J. Lloyd Charitable Foundation, Melanoma Research Alliance Young Investigator Award and Cancer Research

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Institute-CLIP award to P.-C.H.

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The mitochondrion acts as a master regulator to fine-tune immune responses elicited by macrophages and T cells. The Mitochondrion functions as a signaling platform and mitochondrial metabolism are involved in regulation of macrophage activation and functions. Mitochondrial signaling, metabolism and dynamic are crucial for T cell activation, differentiation and function.

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