Reviewing the impact of lipid synthetic flux on Th17 function

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ScienceDirect Reviewing the impact of lipid synthetic flux on Th17 function Yoko Kidani1 and Steven J Bensinger1,2 CD4+ T helper 17 cells (Th17) acquire specific effector functions in response to activation and instructional signals. Accumulating evidence indicates that specific cellular lipid metabolic pathways play essential roles in regulating the differentiation and function of Th17 cells. Mechanistic studies reveal that metabolic fluxes through both the cholesterol and long chain fatty acid biosynthetic pathways are important in controlling RORg transcriptional activity through their ability to generate lipid ligands of RORg. Genetic and pharmacologic studies demonstrate that altering the flux through these lipid biosynthetic pathways impacts the generation of IL-17 as well as the balance of Th17 and CD4+ regulatory T cells (Tregs). In this mini-review, we briefly introduce the mechanics of cholesterol and long chain fatty acid biosynthesis. We also discuss the evidence underlying the unique role that these lipid metabolic pathways play in intrinsically regulating the fate and function of Th17 cells under normal and pathogenic conditions. Addresses 1 Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California, 615 Charles E. Young Drive, Los Angeles, CA 90095, United States 2 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, 615 Charles E. Young Drive, CA 90095, United States Corresponding author: Bensinger, Steven J ([email protected]. edu)

Current Opinion in Immunology 2017, 46:121–126 This review comes from a themed issue on Metabolism of T cells Edited by Jeffrey C Rathmell and Nancie J MacIver

http://dx.doi.org/10.1016/j.coi.2017.03.012 0952-7915/ã 2017 Published by Elsevier Ltd.

is a member of the nuclear receptor superfamily and is responsible for transactivating the signature pro-inflammatory cytokine gene Il17a and Il17f. Accordingly, understanding the signals that directly regulate the expression and activity of RORg remains of significant interest to the infectious disease and rheumatic scientific communities. Unexpectedly, recent studies directly implicate metabolite fluxes through the lipogenic (fatty acid biosynthetic) and cholesterogenic pathways as key regulators of RORg activity under normal and pathogenic conditions. Herein, we briefly describe the metabolic pathways underlying cholesterol and long chain fatty acid biosynthesis and review our current understanding of how these pathways specifically influence the differentiation or function of Th17 cells. Cholesterol and non-essential long chain fatty acids (designated as LCFAs) are critical components of T cells, contributing to their cellular architecture, and a diverse array of cellular processes such as signaling, energy homeostasis, and protein trafficking [5–7]. T cells are endowed with the capacity to synthesize cholesterol and non-essential LCFAs, or import them from their local environment [6,8]. The balance of synthesis and import of these lipids is largely dependent on the activation or functional state of the T cell. In a quiescent state, T cells largely rely on lipid import to maintain lipid and energy homeostasis. The engagement of T cell receptor and activating co-stimulatory molecules (e.g., CD28) rapidly reprograms cellular metabolism to meet the metabolically and energetically demanding requirements of blastogenesis, division and effector function. A significant component of metabolic reprogramming is the upregulation of the cholesterol and lipogenic programs [9,10]. The biochemical details of these critical synthetic pathways are briefly outlined below with an eye towards highlighting enzymes or metabolites that have been specifically implicated in Th17 cell function.

The cholesterol biosynthetic pathway Introduction Th17 cells are a distinct subset of pro-inflammatory T helper CD4 T cells which play an essential role in mediating neutrophilic-inflammatory responses to infectious agents [1,2]. Likewise, the aberrant production of inflammation by Th17 cells has also been shown to be important in driving a number of autoimmune diseases [3]. The master transcriptional regulator of Th17 cells is RAR-related orphan receptor gamma (RORg) [4]. RORg www.sciencedirect.com

Acetyl-CoA is the key metabolite building block of both cholesterol and LCFAs [11,12]. The cytosolic Acetyl-CoA pool is fed by the enzymatic actions of ATP citrate lyase (ACLY), which converts cytosolic citrate into acetyl-CoA and oxaloacetate. In some tissues, acetyl-CoA is also produced by acetyl-CoA synthetase through the ligation of acetate and CoA [13]. Acetyl-CoA is then available for use in both the fatty acid and cholesterol biosynthetic pathways. Canonical de novo cholesterol biosynthesis involves over 20 cytosolic and endoplasmic reticulum Current Opinion in Immunology 2017, 46:121–126

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(ER)-associated enzymes. Although the details of the cholesterol biosynthesis can be perceived as complicated, it is perhaps most useful to divide the elements of the cholesterol biosynthetic pathway into a proximal and distal branch (see Figure 1). In the proximal branch of this pathway (also termed the mevalonate arm), AcetylCoA is converted to squalene through the sequential actions of multiple enzymes, including HMG-CoA reductase (HMGCR), the rate limiting enzyme in this pathway and the pharmacologic target of statins. Importantly, the products of the proximal branch can be shunted into other enzymatic pathways to generate critical metabolites (e.g., ubiquinone, dolichol) or used to generate isoprenoid groups (e.g., isopentenyl pyrophosphate (IPP) and

farnesyl pyrophosphate (FPP)) which are used to modify protein localization and function. The conversion of FPP into squalene, and the subsequent generation of the canonical ring structures associated with sterol metabolites, is considered the distal branch of the cholesterol biosynthetic pathway. Downstream of the intermediate lanosterol, cholesterol biosynthesis divides into two major synthetic pathways, the Kandutcsh-Russel pathway and Bloch pathway. These pathways are primarily distinguished by the timing of the reduction of D24 bond. Defect of each enzyme in these two pathways are known to cause human malformation syndromes [14], indicating the importance of both

Figure 1

Distal branch

Kandutsch-Russell pathway

Bloch pathway

DHCR24 Dihydrolanosterol

Lanosterol

Squalene

CYP51A

Mevalonate

14-demethyl-14-dhydrolanosterol (FF-MAS)

CYP51A DHCR24 Dihydro-FF-MAS

LBR/TM7SF2

LBR/TM7SF2 HMGCR Proximal branch

14-demethyl-lanosterol (T-MAS)

DHCR24 Dihydro-T-MAS

Acetyl-CoA SC4MOL

SC4MOL

NSDHL

NSDHL

ACLY

HSD17B7 Citrate

HSD17B7 DHCR24

Zymosterol

Zymostenol

EBP

EBP

SC5DL

SC5DL

DHCR7

DHCR7 DHCR24 RORY

Desmosterol

Cholestadienol

II17a Current Opinion in Immunology

Cholesterol biosynthesis pathway. The key metabolic intermediates and enzymes in the cholesterol biosynthesis pathway. Metabolite intermediates are outlined in boxes. The cholesterol biosynthetic pathway intermediates and enzymes highlighted in light red have been identified are potential regulators of RORg activity in Th17 cells. Not pictured are oxysterol synthesizing enzymes. ACLY: ATP citrate lyase, HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase, CYP51A: cytochrome P450 family 51 subfamily A, LBR: lamin B receptor, TM7SF2: transmembrane 7 superfamily member 2 (Delta(14)-sterol reductase), SC4MOL: methylsterol monooxygenase 1 (Sterol-C4-mehtyl oxidase-like), NSDHL: NAD(P) dependent steroid dehydrogenase-like, HSD17B7: hydroxysteroid 17-beta dehydrogenase 7, EBP: emopamil binding protein (sterol isomerase), SC5DL: sterol-C5-desaturase, DHCR7: 7dehydrocholesterol reductase, DHCR24: 24-dehydrocholesterol reductase.

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Th17 and lipids Kidani and Bensinger 123

branches on cholesterol homeostasis on development. However, the exact importance of these two distinct branches of cholesterol biosynthetic pathways on T cell biology has not been evaluated. The terminal step in these pathways generates cholesterol which can be subsequently used in cellular processes or further metabolized to cholesterol derivatives such as oxysterols and steroid hormones.

Flux through the cholesterol biosynthetic pathway and RORg activity Recent studies indicate the critical importance of fluxes through the distal branch of the cholesterol biosynthetic pathway in controlling RORg activity, and as such influence RORg dependent thymocyte development and/or CD4 Th17 function [15,1617] (Figure 1). The initial concept of a relationship between sterol metabolites and T cell development/function can be found in the structural analysis of RORg. Several members of the nuclear receptor super family bind to derivatives of cholesterol (e. g., LXR and oxysterols), and their activity is regulated through lipid ligand binding. Mass spectrometry studies focused on determining lipids associated with immunoprecipitated RORg revealed enrichment of sterol metabolites (e.g., oxysterols) and cholesterol biosynthetic intermediates [16]. Mass spectrometry studies also indicated significant increases in the intracellular pool sizes of metabolites from the distal branch of the cholesterol biosynthetic pathway, and specific oxysterols in Th17 cells, when compared to their naı¨ve or Th1 counterparts [15,16,17]. Correspondingly, sterol metabolite and enzyme addback experiments showed that both sterol biosynthetic intermediates and cholesterol-derived metabolites have the ability to increase RORg transcriptional activity [15,16,17]. Loss-of-function studies further support the notion that sterol metabolism influences Th17 cell biology. Genetic deletion of specific enzymes in the distal branch of cholesterol biosynthetic pathway, or mitochondrial sterol metabolism, reduce the production of IL-17 from RORg + CD4 T cells [15,16,17]. Treatment of CD4 T cells with statins, a pharmacologic inhibitor of HMGCR activity, can also impacts the balance of Th17 and Treg cells [18]. As to which specific sterol metabolite serves as the physiologic ligand of Th17 cells in vivo remains to be precisely defined, but they appear to be generated in Th17 cells somewhere after the production of lanosterol in the cholesterol biosynthetic pathway and before cholesterol (Figure 1). Interestingly, both pharmacologic and genetic inhibition of these sterol metabolic pathways were unable to completely ablate IL-17 production [15,16,17], suggesting that other physiologic ligands likely exist. In support of this notion, studies on fatty acid metabolism reveal a potentially important role for fatty acid metabolic pathways on RORg activity www.sciencedirect.com

and Th17 function, the details of which are delineated below.

Fatty acid synthesis Non-essential fatty acids are long chain hydrocarbons which can be produced by the cell, or derived from local environmental sources (e.g., serum and lymph). In general, long chain fatty acids are considered to be between 14 and 20 carbons in length, and can have varying levels of desaturation. Saturated fatty acids (SFA) have no double bonds, monounsaturated fatty acids (MUFA) have one double bond, and polyunsaturated fatty acids (PUFA) have two or more double bonds. The number and position of the double bonds contribute to their biochemical properties (e.g., signaling through the arachidonic acid cascade) or their biophysical properties (e.g., membrane fluidity impacted by packing of phospholipids in the membrane) [19]. Similar to cholesterol, synthesis of LCFAs is reliant on acetyl-CoA as the key building block. Lipogenesis is initiated by the carboxylation of acetylCoA into malonyl-CoA through the enzymatic actions of acetyl-CoA carboxylase (ACC). There are two isoforms of ACC. ACC1 resides in cytosol, and is considered as the rate limiting enzyme for long chain fatty acid synthesis. ACC2 exists on mitochondrial membrane. Control of ACC activity is complex and occurs at the transcriptional and posttranscriptional level. Of particular importance, the upstream metabolite citrate allosterically activates ACC, resulting in heightened flux into the malonylCoA pool when the citrate pool increases. Conversely, changes in energetics results in phosphorylation by AMPkinase and attenuation of ACC activity. Malonyl-CoA can also act as an inhibitor of fatty acid oxidation (FAO) by interfering with the action of carnitine palmitoyltransferase 1 (CPT1), through its ability to block fatty acid entry into the mitochondria. Thus, the pool size of malonylCoA serves as an important regulator of cellular metabolic function by modulating the balance between lipogenesis and beta-oxidation [20]. Acetyl-CoA and malonyl-CoA subsequently undergo condensations and reductions to build long chain fatty acids. This metabolic step is catalyzed by an individual domain of a multi-enzyme protein, fatty acid synthase (FAS). The main product of FAS is 16-carbon palmitic acid, and lauric acid (12-carbon), myristic acid (14-carbon), and stearic acid (18-carbon) to much more limited extent. These saturated fatty acids can subsequently be desaturated by the steroyl-CoA desaturase enzymes. The bulk of these saturated and monounsaturated fatty acids are subsequently incorporated into complex lipids (e.g., phospholipids) for use in membranes, or stored in lipid droplets as triacylglycerides and cholesterol esters for subsequent catabolic processes (e.g., oxidation in mitochondria). To a much more limited, albeit important, extent LCFAs serve as signaling molecules. Current Opinion in Immunology 2017, 46:121–126

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Fatty acid metabolism and Th17 pathogenicity Fluxes through the fatty acid biosynthetic pathway, has also emerged as an important regulator of Th17 plasticity and function (Figure 2). The rate-limiting enzyme ACC1 has been shown to impact that balance of Th17 and Tregs [21]. Pharmacologic and genetic inhibition of ACC1, but not ACC2, attenuated the expression of IL-17 production and increased Foxp3 expression in CD4 T cells cultured under Th17 skewing conditions. This shift towards Treg differentiation in ACC1-deficient cells could be overcome by the addition of exogenous palmitic acid (16:0), suggesting that 16:0 or one of its downstream desaturated or elongated fatty acid products play an important role in entraining Th17 differentiation. Recently, single cell analysis of mice with experimental autoimmune encephalomyelitis (EAE) also implicated the lipogenic pathway in controlling the function of Th17 cells [ 22,23]. RNA-seq studies performed on T cells isolated from EAE mice demonstrated that the CD5 molecule-like (CD5L) was correlated with pathogenicity. CD5L has been shown to regulate flux through the de novo fatty acid biosynthetic pathway by binding to FAS and inhibiting

enzymatic activity [24]. Mechanistic studies revealed that CD5L is highly expressed in ‘non-pathogenic’ Th17 cells when compared to pathogenic Th17 cells counterparts. CD5L-deficient mice develop more severe EAE when compared to wild type controls. And correspondingly, characterization of T cells from these mice showed increased proliferative capacity and IL-17 production. As to how CD5L impacts the pathogenicity of Th17 cells remains an interesting question. Lipidomics study in CD5L-deficient and wild type Th17 cells revealed global differences in fatty acid acyl tail composition of the lipids in both phospholipids and neutral lipids [23]. The known function of CD5L is to inhibit the enzymatic activity of FAS [24], implicating flux through this pathway as the important regulatory difference in complex lipid composition. Analysis of CD5L deficient Th17 cells revealed increased saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) side chains, and a corresponding decrease in polyunsaturated fatty acid (PUFA) side chain such as C20:4 [23]. Moreover, the exogenous addition of PUFA to pathogenic Th17 cells decreased RORg binding at Il17 and Il23r loci, whereas providing

Figure 2

Citrate ACLY

ACC1

Malonyl-CoA

Acetyl-CoA

ACC2 FAS Malonyl-CoA

Long chain fatty acid (SFA) SCD, ELOVL MUFA

CPT1A

FADS, ELOVL PUFA

Mitochondrion

RORY II17a Current Opinion in Immunology

Fatty acid biosynthesis pathway. A schematic view of fatty acid synthesis pathway with the key metabolic intermediates and enzymes. Metabolite intermediates are outlined in boxes. The fatty acid synthetic pathway intermediates and enzymes highlighted in light red have been identified are potential regulators of RORg activity in Th17 cells. ACC: acetyl-CoA carboxylase, FAS: fatty acid synthase, SCD: stearoyl-CoA desaturase, ELOVL: fatty acid elongase, FADS: fatty acid desaturase. SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids. Current Opinion in Immunology 2017, 46:121–126

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Th17 and lipids Kidani and Bensinger 125

exogenous SFA increased RORg occupancy [23]. Thus, the intracellular balance of these fatty acid populations appears to impact Th17 function at least in part via RORg recruitment and/or activity at the signature cytokines. While it is not entirely clear what the molecular mechanisms linking fatty acid homeostasis and RORg function, perhaps the most parsimonious explanation is that specific fatty acid containing lipids can also bind to, and regulate RORg activation in a manner similar to cholesterol biosynthetic intermediates. Alternatively, it may be that balance of fatty acid saturation and desaturation regulates flux through the cholesterol biosynthetic pathway, thereby impacting the availability of sterol derived ligands.

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Concluding remarks

11. Auchus RJ, Adams CM: Cholesterol, Steroid and Isoprenoid Biosynthesis. Chichester: eLS. John Wiley & Sons Ltd; 2005.

It is now clear that cholesterol and fatty acid homeostasis intrinsically impacts the fate and function of Th17 cells. However, significant gaps in our knowledge about lipids in Th17 cells still remain. For example, it remains unclear as to why fluxes through these lipid biosynthetic pathways would specifically regulate the function of RORg and production of their signature cytokines. Given that these lipid metabolic pathways are critical for the anabolic processes of nearly all proliferating T cells, it remains a mystery as to why lipid metabolism should be co-opted to regulate RORg function in Th17 cells. Perhaps, identifying the exact physiologic lipid ligands of RORg will afford insights as to how lipid metabolism specifically influences Th17 function, and ‘why’ the cross-talk between cholesterol or fatty acid homeostasis and Th17 cells has evolved. Finally, the significant insights gained over the last few years strongly indicate that targeting lipid homeostasis in T cells could serve as a valuable therapeutic tool for restraining the pathogenicity of Th17 cells, reshaping the balance of Th17 and Tregs underlying disease conditions.

Acknowledgement This work was supported by NIHAI093768 and HL126556 (to S.J.B.).

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of outstanding interest 1.

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