The Metabolic Signature of T cells in Rheumatoid Arthritis

Abstract

Purpose of review:

Rheumatoid arthritis (RA) is a prototypic autoimmune disease manifesting as chronic inflammation of the synovium and leading to acceleration of cardiovascular disease and shortening of life expectancy. The basic defect causing autoimmunity has remained elusive, but recent insights have challenged the notion that autoantigen is the core driver.

Recent findings:

Emerging data have added metabolic cues involved in the proper maintenance and activation of immune cells as pathogenic regulators. Specifically, studies have unveiled metabolic pathways that enforce T cell fate decisions promoting tissue inflammation; including T cell tissue invasiveness, T cell cytokine release, T cell-dependent macrophage activation and inflammatory T cell death. At the center of the metabolic abnormalities lies the mitochondria, which is consistently underperforming in RA T cells. The mitochondrial defect results at least partially from insufficient DNA repair and leads to lipid droplet accumulation, formation of invasive membrane ruffles, inflammasome activation and pyroptotic T cell death.

Summary:

T cells in RA patients, even naïve T cells never involved in inflammatory lesions, have a unique metabolic signature and the changes in intracellular metabolites drive pathogenic T cell behavior. Recognizing the role of metabolic signals in cell fate decisions opens the possibility for immunomodulation long before the end stage synovial inflammation encountered in clinical practice.

Introduction

Rheumatoid arthritis is diagnosed in a host with chronic symmetrical and erosive polyarthritis and a spectrum of autoantibodies reactive against the Fc region of IgG, cartilage components, nuclear proteins and an array of citrullinated proteins [1, 2]. Such autoantibodies, indicative of a fundamental breakdown in self-tolerance, are present decades before clinical symptoms. How this breakdown comes about has remained unresolved. Traditional paradigms have focused on access to autoantigen, but the broad spectrum of autoantigens seen by the RA host favors systemic abnormalities in immune homeostasis. Specifically, HLA class II restricted CD4 T cells, indispensable to induce and sustain autoantibody production by B cells, are key elements in the pathogenic pathways characteristic for RA [3–6]: invasion of synovial membrane by innate and adaptive immune cells, activation of synovial stromal cells, formation of organized lymphoid architectures in the joint, stimulation of bone-erosive osteoclasts, induction of atherosclerotic plaque, etc. Since autoantibody production precedes synovitis by decades, T cells must have abnormalities long before RA is diagnosed. To examine fundamental tolerance defects in T cells one has two choices: study such cells in individuals prior to disease onset or isolate out of RA patients the naïve T cell population. Such naïve T cells live in lymph nodes and the bone marrow, have never been engaged in an inflammatory lesion and represent the “T cell reserve”, called into action when antigen is being recognized to then clonally expand and differentiate into dedicated memory and effector cells.

Work from the last 5 years has demonstrated that naïve CD4 T cells isolated from RA patients are clearly distinct from their counterparts in healthy age-matched individuals. The distinguishing features include a growing list of molecules that are under- or overexpressed in RA T cells. Most of the molecules abnormally expressed in RA T cells fall into the category of metabolic regulators [7–11]; ranging from metabolic enzymes to metabolites and essential parts of metabolically active organelles. A surprising finding has been the role of mitochondria in RA T cells [12, 13]. While well established as the powerplant of cells, mitochondria are now emerging as sensors of cellular stress, integrators of activating signals, stress signals and metabolic cues and facilitators of cell fate decision and cell survival [14–16]. Here, we will review recent work on the role of T cell mitochondria in synovial inflammation.

T cells from RA patients age prematurely and accumulate DNA damage

More than a decade ago, the observation was made that naïve CD4 T cells isolated from RA patients have age-inappropriate shortening of telomeres [17, 18]. As telomeres serve as the sensors of cellular age, this observation gave rise to the concept that RA T cells are prematurely aged. As a general rule, aging of T cells is associated with functional decline and the appearance of receptor and ligands typically encountered on NK cells [10, 19]. Thus, old T cells are less competent and less precise and acquire features of innate cells, biasing them towards inflammatory effector functions. T cell aging is also coupled to a fundamental change in cellular metabolism [20, 21]. A hallmark of aged cells is the inability to fully mobilize mitochondria for ATP synthesis and provision of metabolic intermediates that are formed in the tricarboxylic acid (TCA) cycle. Recent studies [12] have identified mitochondria in RA T cells as being low in oxygen consumption and ATP production, suggestive for defects in both the electron transport chain and in oxidative phosphorylation.

Several molecules have been identified that play a role in the prematurity of T cell aging in RA patients: telomerase, the DNA repair kinases ATM, DNA-PKcs and the DNA repair nuclease MRE11A [22]. Besides their role in sensing and repairing broken DNA, ATM, DNA-PKcs and MRE11a, all are involved in regulating cellular metabolism and are interconnected with mitochondrial fitness and energy generation pathways (Table 1). ATM promotes cell cycle arrest to accomplish DNA double strand break repair. ATM responds to reactive oxygen species (ROS) by forming homodimers that detect DNA damage and pause the cell cycle. Intracellular ROS levels are a direct reflection of metabolic status and mitochondrial activity, linking ATM activation to periods of high energy generation. ATM is distinctly low in RA T cells [23], in line with impairment of mitochondrial fitness. ATM deficiency accelerates synovial inflammation, possible through the kinase’s involvement in cell cycle regulation. In contrast, the repair kinase DNA-PKcs is disproportionally high in RA T cell [24]. In line with direct involvement of DNA-PKcs in suppressing mitochondrial activity [25] and promoting de novo lipogenesis [26, 27], RA T cells have been reported to be biased towards fatty acid synthesis and deposition of lipid droplets [28]. The final molecule actively engaged in DNA damage sensing and repair as well as in metabolic regulation is MRE11A, a protein with 3′ to 5′ exonuclease activity as well as endonuclease activity [29, 30]. The concentration of MRE11A protein in naïve and memory T cells declines with age [31], making the nuclease a sensitive marker of immune aging. Aging-related decline of T cell MRE11A is accelerated in RA T cells, in line with prematurity of T cell aging. MRE11A localizes to the telomere and loss-of-function causes several structural defects [31], including fragility of chromosomal ends. Thus, age-inappropriate erosion of telomeric sequences in RA T cells, first described two decades ago, may be a consequence of insufficient DNA repair.

Table 1.

DNA repair defects and metabolic dysregulation in T cells from RA patients

Molecule	RA T cells (compared to normal T cells)	Role in DNA repair	Role in metabolism
ATM (Ataxia Telangiectasia Mutated)	Low expression	Repair of double-strand DNA breaks by homologous recombination; cell cycle arrest	Sensor of (mitochondrial) reactive oxygen species
DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)	High expression	Repair of double-strand DNA breaks by non-homologous end joining (NHEJ)	Suppresses mitochondrial function Promotes fatty acid synthesis
MRE11A (Meiotic Recombination 11 Homolog)	Low expression	DNA endo- and exonuclease Telomere maintenance	Mitochondrial fitness

T cells from RA patients have a defect in mitochondrial DNA repair and leak mtDNA into the cytoplasm

MRE11A loss-of-function not only is mechanistically connected to genome instability at the telomeric ends; more importantly, MRE11A^low T cells have a phenotype of mitochondrial failure [12, 31]. Under basal and stressed conditions, MRE11A^low mitochondria consume low amounts of oxygen, indicating malfunction of the electron transport chain. T cells rendered MRE11A^low and RA T cells, which are spontaneously low in MRE11A, share distinctly low production of ATP. Biochemical and imaging studies have placed MRE11A into the mitochondrial matrix, where it binds to mitochondrial DNA. MRE11A deficiency exposes mitochondrial DNA to oxidative attack and leads to leakage of mtDNA into the cytoplasm (Figure 1). Leaked mtDNA is detected by the AIM2 and the NLRP3 inflammasome and triggers activation of caspase-1. The most important consequence of this process is the lytic death of the MRE11A-unprotected RA T cells. Caspase-1 activation was demonstrated in the lymph nodes of RA patients, in line with the systemic nature of the T cell defect. Indeed, in vivo experiments demonstrated the deposition of mtDNA in inflamed synovial tissue, supporting the novel concept that tissue-infiltrating T cells drive inflammation through their lysis and not through their presence. This novel paradigm focusses attention away from the tissue lesions to T cell fate decisions in the secondary lymphoid organs.

Figure 1. Mitochondrial Defects in RA T cells.

Besides being part of the nuclear DNA repair machinery, the DNA nuclease MRE11A also localizes to the mitochondrial matrix where it binds to mitochondrial DNA (mtDNA). MRE11A protects mtDNA from damage and controls mtDNA containment in the organelle. In RA T cells, which express a low amount of mitochondrial MRE11A, mtDNA leaks into the cytoplasm to trigger assembly of the AIM2 and NLRP3 inflammasome and activation of caspase-1. As a result, T cells release IL-1b and IL-18 and undergo lytic death (T cell pyroptosis); culminating in aggressive tissue inflammation. MRE11A; meiotic recombination 11. AIM2; absent in melanoma 2. NLRP3; NLR Family Pyrin Domain Containing 3. IL-1b; Interleukin 1 beta. IL-18; Interleukin 18.

T cells from RA patients deviate glucose away from glycolysis into the pentose phosphate pathway

Careful studies have uncovered metabolic defects in RA patients and have linked them to pathogenic behavior in in vivo model systems, making a direct connection between metabolic wiring and inflammation-inducing effector functions [28, 32–34]. In contrast to most other somatic cells, T cells have enormous proliferation capacity and grow massively following antigen recognition. The generation of clonal offspring is associated with high biosynthetic and energy demand, required to build the membranes, organelles, DNA and RNA for the daughter cells. Thus, T cell growth and survival is inevitably coupled to the metabolic machinery [35–37].

Naïve T cells preferentially rely on fatty acid oxidation [38–40], an energy generation state strictly linked to intact mitochondria. Immediately following T cell activation, both glucose and the amino acid glutamine are major energy carriers and the cell upregulates glycolytic as well as glutaminolytic activity [41, 42]. Effector T cells, including T cells entering the joint to function as multipotent amplifiers of tissue inflammation, remain dependent on fast access to glucose and generate ATP through glycolysis. Thus, fate decisions that enforce activation and effector differentiation of T cells depend on glucose and glutamine-rich microenvironments [43]. In contrast, triggering of memory T cell differentiation is accompanied by return of the T cell to fatty acid oxidation. Taken together, maintenance of naïve and memory T cells is closely linked to mitochondrial fitness [44], whereas effector T cells, including the pro-inflammatory T cells that sustain inflamed tissue sites, fuel their energy demands through fast access to glucose and amino acids.

RA T cells, as discussed above, have restrictions in activating mitochondria [12]. Due to ineffective DNA repair, basic mitochondrial functions are impaired, rendering the cells reliant on extra-mitochondrial energy production. RA T cells have rewired their metabolic program such that they can function as “supereffector cells” (Figure 2), ready to leave lymphoid tissues and invade into peripheral tissue niches, such as the synovial membrane. Once in the tissue, they have a low threshold to unload effector cytokines and activate surrounding stromal cells, such as endothelial cells and synovial fibroblasts. A major inflammatory trigger derives from the pyroptotic cell death of T cells with leaky mitochondria; releasing intracellular content, including DNA into the extracellular space to function as a danger-associated molecular pattern.

Figure 2. Metabolic Programs in healthy and RA T cells.

Depending on their state of differentiation, T cells preferentially use fatty acid oxidation, glycolysis, glutaminolysis or the pentose phosphate pathways to satisfy their energy needs and their demand for biosynthetic precursors. Metabolic preference makes them dependent on mitochondrial fitness. The dominant metabolic programs are compared in healthy T cell subsets and in RA effector T cells. PPP; pentose phosphate pathway.

The metabolic reprogramming of RA T cells is characterized by the following molecular events:

• Transcriptional repression of phosphofructokinase (PFK), leading to suppression of glycolytic breakdown [34], reduced pyruvate production and low secretion of lactate into the extracellular milieu.

• Transcriptional upregulation of glucose-6-phosphate dehydrogenase (G6PD), resulting in enhanced activity of the pentose phosphate pathway (PPP), producing excess amounts of NADPH and reduced glutathione. The shunting of glucose to the PPP creates a reductive environment [8, 11].

It is currently unclear what the upstream signals are that impose the redirection of glucose from glycolysis to PPP. Downstream consequences include lack of pyruvate to supply the mitochondria, excess NADPH production, lower ROS concentrations and failed activation of ATM [7, 33]. Isolated knockdown of PFKFB3 is sufficient to produce a pro-inflammatory T cell phenotype and generate robust synovial inflammation in vivo. Similarly, treating with oxidizing agents in vivo has strong anti-inflammatory effects, providing evidence that metabolic interference in T cells alone is sufficient to regulate susceptibility or resistance to tissue inflammation.

T cells from RA patients store lipid droplets and form invasive membrane structures

Besides the electron transport chain, mitochondria also house the tricarboxylic acid (TCA) cycle, a series of chemical reactions in which the acetyl group is oxidized to carbon dioxide and water, while ATP and GTP are produced as energy storage molecules. The acetyl group arrives in the mitochondria as acetyl-CoA, mostly when pyruvate undergoes oxidative decarboxylation. Low input of pyruvate will result in low acetyl-CoA production and slowing down of the TCA cycle. Instead, acetyl-CoA is transported into the cytoplasm (in the form of citrate) where it is used to synthesize fatty acids through the action of acetyl-CoA carboxylase [45]. In parallel, mitochondrial dysfunction will disrupt β-oxidation, the major breakdown mechanism for fatty acids. As an end result, RA T cells have oversupply in fatty acids. Appropriate biochemical studies have confirmed that RA T cells have upregulated the lipogenesis program [28] and produce fatty acids instead of utilizing acetyl-CoA as a source of ATP generation. Fatty acid synthesis is further facilitated by the availability of NADPH (Figure 3).

Figure 3. Energy rerouting towards biosynthesis and lipogenesis in RA T cells.

Glucose is a major energy carrier for T cells, providing ATP and biosynthetic precursors. In RA T cells, glycolytic breakdown is disfavored, impairing ATP generation. Instead, RA T cells shunt glucose into the pentose phosphate pathway to generate NADPH, biosynthetic precursor, and eventually fatty acids. The shift towards lipogenesis results in the deposition of lipid droplets in the cytoplasm and the rapid access to membrane lipids, rendering the T cells tissue invasive.

ATP; adenosine triphosphate. NADPH; reduced form of nicotinamide adenine dinucleotide phosphate.

RA T cells adopt to the excess fatty acids by depositing lipid droplets in their cytoplasm. Ample lipid storage enables such T cells to rapidly utilize fatty acids for the fabrication of membrane bilayers, a necessary pathway in the cell building program. T cells residing in synovial tissue of RA patients carry cytoplasmic lipid droplets, a feature previously associated with the metabolic adaptations of tumor cells. An active membrane building program has been identified in RA T cells through co-localization studies of the cytoskeletal marker F-actin and the membrane marker cortactin [46, 47]. The most valuable data from these studies demonstrated that in lipid-droplet containing T cells membranes were arranged in invasive membrane ruffles, reminiscent of podosomes utilized by tumor cells to spread and metastasize [48]. Functionally, membrane ruffling promoted T cell migration in extracellular matrix and in tissue.

It is not surprising that the T cell motility machinery is integrated with metabolic cues, as supply of ATP and biosynthetic molecules is a prerequisite of T cell migration and tissue invasion. The molecular signature of the membrane-building program in RA T cells is anchored around the adaptor protein Tks5 (encoded by the SH3PXD2A gene). Tks5 acts as an organizer at the plasma membrane [49]. RA T cells typically express higher Tks5 concentration than matched control T cells. Loss-of-function (knockdown) experiments and gain-of-function (overexpression) experiments have demonstrated that Tks5 is a critical regulator of T cell behavior. Tks5 gain is sufficient to render T cells tissue invasive and inflammatory. Tsk5 loss can correct this phenotype and suppress synovial inflammation.

Lysosomal abnormalities in RA T cells

Dysfunctional mitochondria generating insufficient ROS and ATP and failing to burn fatty acids, are a key metabolic defect in RA T cells. Another subcellular organelle that contributes to the altered metabolic state of RA T cells is the lysosome [32, 50–52] (Figure 4).

Figure 4. Altered protein trafficking and defective lysosomal AMPK recruitment in RA T cells.

The energy sensor AMPK needs to be recruited to the lysosomal surface to co-localize with the kinase mTOR, which coordinates cell growth and differentiation with environmental conditions, including energy availability. AMPK is triggered by accumulation of AMP (and ATP reduction) and inhibits activation of mTORC1. Conversely, surplus of ATP inactivates AMPK and stimulates mTORC1 to facilitate T cell growth, differentiation, and migration. Recruitment of AMPK to the lysosome requires membrane anchoring through a lipid tail, which is attached by the enzyme N-myristoyltransferase (NMT). In RA T cells, activity of NMT1 is distinctly low, leaving AMPK without a lipid tail and preventing lysosomal recruitment. mTORC1 is unopposed and drives anabolic metabolism, cellular expansion, and T cell motility.

AMPK; AMP-activated protein kinase. mTORC1; mechanistic target of rapamycin complex 1. NMT1; N-Myristoyltransferase 1

As long-lived cells that need to persist in harsh tissue environments, T cells sense environmental cues and couple them to metabolic activity and cell growth. A key integrator is the kinase mTOR, which under steady-state conditions is suppressed by multiple inhibitory mechanisms [37, 53, 54]. Antigen recognition triggers activation of mTOR, which orchestrates the differentiation of T cells into functional lineages, and thus determines their role in chronic inflammatory disease. Emerging data have implicated mTOR as a signaling node to regulate metabolic conditioning of T cells and their migratory activity. A general rule is that pro-inflammatory effector cells switch on a highly anabolic program, with high consumption of glucose and glutamine and concomitant suppression of fatty acid oxidation. Here, mTOR is critically involved in suppressing AMPK signaling and thus a major driver of mitochondrial biogenesis and function. Vice versa, catabolic conditions, favored by regulatory and naïve T cells, rely on energy production in the mitochondria, utilizing oxidative phosphorylation as well as beta-oxidation. The independence of RA T cells from mitochondrial activity is in line with a super-anabolic state.

In RA T cells, mTORC1 is strongly activated [32], enabling the cell to build progeny, migrate to inflammatory sites and produce an array of pro-inflammatory cytokines. Recent studies have attributed unopposed mTORC1 activation to a failure in the energy sensor AMPK. Despite low cellular concentrations of ATP and a shift in the AMP/ATP ratio towards energy deprivation, RA T cells fail to activate AMPK. This failure was linked to misplacement of AMPK within the cell, precisely, failed recruitment of AMPK to the cytoplasmic face of the lysosome [32, 51]. On the lysosomal surface, AMPK and mTORC1 come together and counter-regulate each other [55–57]. Energy starvation leads to AMPK activation and mTORC1 repression, blocking T cell from growth and expansion. Conversely, energy surplus triggers inactivation of AMPK, resulting in mTORC1 activation and preparation of the T cell for an expansion program. The underlying defect in RA T cells has been attributed to a defective lysosomal recruitment mechanism. Localization of AMPK to the lysosomal surface requires membranous anchoring, which is achieved through the covalent attachment of a C14-fatty acid tale to the protein. Attachment of myristic acid to AMPK is catalyzed by the enzyme N-myristoyltransferase (NMT) [58, 59] and studies in patients with several autoimmune diseases have demonstrated that RA patients have a selective suppression of NMT1. The functional defect of NMT1 in RA T cells leads to subcellular redistribution of AMPK, which no longer accumulates on the lysosomal membrane. Loss-of-function and gain-of-function experiments confirmed the dependence of mTORC1 activation in RA T cells on lysosomal placement of AMPK and coupled the failure in intracellular protein trafficking to synovial tissue inflammation. Thus, cellular organelles, such as mitochondria and lysosomes, appear to play a critical role in maintaining tissue tolerance and protecting tissue from inflammatory attack.

Conclusion

Chronic tissue inflammation, such as rheumatoid synovitis, is dependent on innate [60] and adaptive [61, 62] immune cells and tissue-residing stromal cells [63]. T cells regulate the activity of innate and stromal cells and, due to their ability to memorize, are a key driver of disease chronicity. T cell function and fate is ultimately determined by their handling of energy sources and their intracellular metabolism. RA T cells display a metabolic signature that distinguishes them from healthy T cells and the RA metabolic program fuels cellular behavior that fosters tissue inflammation. Metabolic reprogramming has also been described for monocytes and macrophages in RA patients [64], but metabolic signatures are cell type specific, compatible with a role of cell endogenous programs.

The metabolic defect in RA T cells centers on the mitochondria and on lysosomes. Precisely, mitochondrial infidelity in RA T cells gives rise to poor ATP and ROS production and suppresses beta-oxidation of fatty acids. The outcome is a reductive cellular environment in which fatty acids are stored in lipid droplets and utilized for the building of invasive membrane ruffles. T cells become hyperproliferative and tissue invasive. Once in the tissue, they are prone to undergo pyroptotic death [65, 66], a highly inflammatory death pathway triggered by the inflammasome recognizing DNA leaking out of the dysfunctional mitochondria. T cell pyroptosis results in the tissue deposition of inflammatory molecules and promotes multiple pathways of chronic-destructive tissue inflammation.

Key points.

• CD4 T cells from patients with RA shift energy carriers, such as glucose from catabolic to anabolic pathways to support a cell building program.

• Due to insufficient DNA repair, mitochondria in RA T cells are defective, leading to leakage of mitochondrial DNA, triggering of the inflammasome and pyroptotic death.

• RA T cells fail to recruit the energy sensor AMPK to the surface of lysosomes, promoting unopposed activation of mTOR and unrestricted cellular expansion.

• RA T cells with defective mitochondria and altered trafficking of AMPK change their fate decisions and behavior and favor hyperproliferation, tissue invasion and pro-inflammatory effector functions.