Nrf2 as a Therapy Target for Th17-Dependent Autoimmune Disease

Abstract

Th17 cells are a subset of IL-17-expressing CD4⁺ T helper cells and play a predominant role in the pathogenesis of autoimmune diseases such as multiple sclerosis and psoriasis. Th17 cells sustain their activation and effector functions primarily through a metabolic profile characterized by high glycolytic and oxidative metabolism. Both glycolysis and OXPHOs can affect cellular redox status, and vice versa. Nrf2, a master regulator of redox homeostasis, plays a pivotal role in oxidative stress regulation and influences immune cell function. This chapter summarizes the recent advances in the understanding of redox regulation in Th17 cells and explores the therapeutic potential of targeting Nrf2 in Th17-dependent autoimmune diseases. Overall, targeting Nrf2 holds considerable promise as a novel therapeutic paradigm for Th17-dependent autoimmune diseases, offering new avenues for precision medicine and improved disease outcomes.

1. Introduction

Autoimmune diseases, characterized by immune system dysfunction and self-directed attacks on healthy tissues, represent a significant challenge in clinical medicine. Among the various immune cell subsets implicated in autoimmunity, Th17 cells have emerged as key players due to their potent pro-inflammatory properties [1]. Th17 cells represent a distinct subset of CD4⁺ T lymphocytes characterized by their production of interleukin-17 (IL-17) cytokines. Originally identified for their role in host defense against extracellular pathogens, mounting evidence implicates dysregulated Th17 cell responses in the pathogenesis of autoimmune disorders, including multiple sclerosis (MS), rheumatoid arthritis (RA), and psoriasis [1]. Their pro-inflammatory properties contribute to tissue inflammation, damage, and chronicity, highlighting the importance of understanding the regulation and function of Th17 cells in health and disease [2]. Thus, there is an imperative clinical demand for therapies capable of efficiently managing Th17-mediated autoimmunity.

Studies have shown that Th17 cells utilize both glycolysis and oxidative phosphorylation (OXPHOs) as energy sources [3, 4]. During the glycolysis and OXPHOs, reactive oxygen species (ROS) are produced as by-products [5]. Given the high metabolic activity of differentiated Th17 cells, there may be more production of ROS during Th17 differentiation. This heightened ROS production can perturb cellular redox balance and potentially lead to oxidative stress within the cellular milieu [6]. ROS-mediated oxidative damage to biomolecules such as lipids, proteins, and DNA can exacerbate tissue inflammation and further contribute to Th17-mediated immunity and the development of autoimmune diseases [7].

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor renowned for its pivotal role in cellular defense mechanisms. Nrf2 orchestrates the expression of a multitude of genes involved in antioxidant response, detoxification, and cellular metabolism, thereby safeguarding cells from oxidative damage and maintaining redox homeostasis [8]. Emerging evidence suggests that Nrf2 exerts profound effects on immune cell function [7]. Various immune cell subsets, including macrophages, dendritic cells, T cells, and natural killer cells, have been shown to express Nrf2 and exhibit altered phenotypes upon its modulation. This modulation extends beyond traditional antioxidant and detoxification pathways, implicating Nrf2 in the regulation of immune cell development, activation, differentiation, and effector functions [9].

Unraveling the intricate interplay between Th17 cell differentiation, metabolic pathways, and cellular redox status has revealed promising therapeutic avenues. By modulating Nrf2 activity, either pharmacologically or through dietary interventions, it becomes feasible to regulate Th17 cell differentiation and function, thereby mitigating autoimmune pathology. This study will discuss evidence of Nrf2-mediated modulation of Th17 cells and underscore recent studies supporting the efficacy of Nrf2 activation as a therapeutic approach. Furthermore, it provides insights into Nrf2 as a potential therapy target for Th17-dependent autoimmune disease, offering a perspective on the development of novel treatment approaches in this context.

2. Metabolic regulation of Th17 cells

Th17 cells represent a distinct subset of CD4⁺ T lymphocytes characterized by their production of IL-17 cytokines. Th17 cells have both non-pathogenic and pathogenic features. The non-pathogenic aspect helps to protect the host against external bacterial and fungal pathogens [10], while the pathogenic facet is involved in driving the progression of autoimmune diseases such as MS, RA, and psoriasis [1]. Compared to the quiescent and metabolically inactive naïve CD4⁺ T cells, Th17 cells are metabolically active [4]. Th17 cells exhibit distinct metabolic profiles, with metabolic reprogramming essential for their differentiation, activation, and effector functions. The metabolic demands of Th17 cells vary depending on the context of activation. Homeostatic Th17 cells induced by commensal bacteria, such as segmented filamentous bacteria, primarily rely on OXPHOs [11]. In contrast, pro-inflammatory Th17 cells elicited in response to pathogens like Citrobacter rodentium exhibit high glycolytic activity [11]. Metabolic intermediates and signaling pathways not only fuel Th17 cell differentiation but also modulate cytokine production such as IL-17, IL-21, IL-22, and GM-CSF, thereby influencing autoimmune disease pathogenesis. Targeting metabolic pathways in Th17 cells offers promising therapeutic strategies to modulate autoimmune inflammation and restore immune balance. Although other metabolic processes such as lipid metabolism and amino acid metabolism are also involved in Th17 differentiation and serve as therapeutic targets for Th17-mediated autoimmune diseases [4], the current study mainly focuses on glycolysis and OXPHOs metabolic regulation in Th17 cells, summarizing their potential therapeutic strategies for Th17-related autoimmune diseases.

2.1 Glycolysis in Th17 differentiation

Glycolysis serves as the primary energy source in differentiated Th17 cells. During cell proliferation and differentiation, large amounts of glucose are transported into the Th17 cell to produce energy for various physiological processes. High glucose levels have been demonstrated to promote Th17 differentiation, exacerbating autoimmunity in mouse models [12]. The significance of glycolysis in Th17 cells has been underscored by numerous studies revealing dysregulated effector functions upon inhibition of glycolytic enzymes’ activities or intermediate products. Genetic deficiency or pharmacological inhibition of key glycolytic molecules, such as hypoxia-inducible factor 1α (Hif1α), glucose transporter (GLUT), pyruvate kinase isoform 2 (PKM2), and pyruvate dehydrogenase kinase 1 (PDHK1), leads to significant impairments in Th17 cell differentiation and pro-inflammatory function [13, 14, 15, 16, 17].

HIF1α plays a crucial role in directing the differentiation of Th17 cells by directly activating the transcription of the Th17 master regulator RAR-related orphan receptor gamma (RORγt). Mice with T cells deficient in HIF1α demonstrate resistance to the induction of Th17-dependent experimental autoimmune encephalitis (EAE), a mouse model of autoimmune disease characterized by decreased Th17 cells and increased Treg cells [13]. In addition, the increased glycolytic rates in Th17 cells coincide with elevated expression of GLUT1, and increased GLUT1 levels are associated with elevated release of IL-17 and IFNγ [14]. Another glucose transporter protein, GLUT3, is also reported to be essential for both the differentiation and effector function of Th17 cells, as evidenced by the protection of GLUT3 knockout mice against EAE [15]. Furthermore, inhibition of PKM2 by vitamin B5 disrupts PKM2 phosphorylation and nuclear translocation, resulting in the inhibition of glycolysis and impaired Th17 cell differentiation. This inhibition leads to protection against EAE [16]. Moreover, PDHK1, a kinase that inhibits pyruvate dehydrogenase to suppress pyruvate oxidation and instead promotes pyruvate conversion to lactate, is essential for Th17 effector function. Blocking of PDHK1 with dichloroacetate suppresses glycolysis, Th17 differentiation, and attenuates EAE development [17].

Together, these findings highlight the critical role of glycolysis in regulation of Th17 cells and suggest that targeting glycolysis could offer therapeutic promise for alleviating Th17-based immune disorders.

2.2 OXPHOs in Th17 cell differentiation

Glycolysis and OXPHOs are two primary metabolic pathways responsible for generating energy within cells. Glycolysis fuels the tricarboxylic acid cycle (TCA) cycle and supports OXHPOs by generating pyruvate. The pyruvate generated from glycolysis is imported into the mitochondria and converted to acetyl-CoA. Acetyl-CoA then enters the TCA cycle to produce NADH and FADH2, which are utilized for generating ATP via OXPHOs. Conversely, OXPHOs can affect glycolysis by influencing metabolic intermediates (e.g., citrate) and cellular signaling pathways (e.g., AMPK signaling) [18, 19]. Thus, the two pathways are interconnected, working in concert to maintain cellular energy balance.

While glycolysis was thought to dominate Th17 cell metabolism, research has also highlighted the importance of OXPHOs in sustaining Th17 cell responses [20]. Studies have indicated that Th17 cells rely on OXPHOs for their effector responses [21]. Consistent with this observation, proliferating Th17 cells have been shown to rely on both electron transport chain (ETC) complex I and complex II activation for IL-17 production [22]. Additionally, estrogen receptor alpha signaling has been implicated in enhancing pathogenic Th17 cell differentiation by promoting ETC complex IV assembly and preserving OXPHOs [23]. In support of these studies, inhibiting OXPHOs has been found to effectively alleviate symptoms of colitis and psoriasis in murine models [21]. Furthermore, treatment with oligomycin, an ETC complex V inhibitor, impaired glycolytic functions and the TCA cycle in Th17 cells, resulting in reduced expression of IL-17, IL-23, and Tgfβ3. Importantly, oligomycin-treated Th17 cells exhibited reduced EAE severity upon adoptive transfer in vivo [20]. Interestingly, oligomycin treatment also promoted the generation of Treg cells via upregulating expression of IL-10 receptor [20].

Taken together, mitochondrial functions and OXPHOs contribute to Th17 cell generation. However, it is noteworthy that, compared to studies focusing on glycolysis, there is a relatively limited amount of research investigating the role of OXPHOs in Th17 cell differentiation and function. Thus, more studies are required to fully elucidate the impact of OXPHOs on Th17 cells and their contribution to immune responses.

3. Interplay between ROS, metabolic flux, and Th17-mediated autoimmunity

ROS are highly unstable, oxygen-containing molecules. ROS encompasses superoxide anion radical, hydroxyl radical, and hydroperoxyl radical [7]. ROS are produced from various sources, and one of the most significant sources is OXPHOs. Upon TCR activation, calcium ions exit from endoplasmic reticulum and influx into mitochondria to promote TCA cycle activity and increase mitochondrial function [5]. Increased TCA cycle leads to increased production of redox coenzymes NADH and FADH2. NADH and FADH2 are the main drivers of ETC by delivering electrons to the ETC in mitochondria. As electrons move through mitochondrial complexes, some may inadvertently leak, leading to the generation of superoxide radicals. In cells with high metabolic states, such as proliferating T cells, more superoxide radicals can be produced, culminating in uncontrolled oxidative stress [6]. Such uncontrolled oxidative stress has been associated with the development of various human diseases, including autoimmune disorders [7].

The association between a high glycolysis flux and increased ROS levels has been established [24]. This association arises from the fact that an increase in glycolytic flux typically leads to a rise in TCA cycle flux. Consequently, there is an accumulation of substrates (e.g., NADH and FADH2) for OXPHOs, potentially stimulating superoxide production [25]. Consistently, in T cells, ADP-dependent glucokinase has been identified as a mediator connecting high glycolysis with production of ROS, elucidating the critical role of glycolytic enzymes in modulating ROS levels [26]. Inhibition of mitochondrial pyruvate uptake restored mitochondrial ROS production to normal levels, thereby blocking the hyperglycemic damage [27]. Moreover, under hyperglycemic conditions, ROS production is induced through the process of mitochondrial fission [28]. These findings collectively demonstrate that increased glycolysis indirectly contributes to ROS and mitochondrial dysfunction. Conversely, ROS can impact glycolysis, forming a feedback loop by stabilizing HIF1α and promoting cell proliferation [29]. Stabilized HIF1α further facilitates a metabolic shift favoring anaerobic glycolysis and reducing mitochondrial respiration by upregulating GLUTs and glycolytic enzymes [30]. Thus, there is an intricate interplay between glycolysis and ROS regulation.

Studies have explored the role of ROS in the differentiation and pathogenicity of Th17 cells. Specifically, deficiency in NADPH oxidase, which maintains low mitochondrial ROS levels, has been found to enhance Th17 differentiation and result in increased susceptibility to EAE [31]. Additionally, knockout of IEX-1 gene, which suppresses mitochondrial ROS production, leads to enhanced Th17 differentiation and exacerbation of arthritis symptoms in a mouse model [32]. Moreover, high glucose concentrations have been demonstrated to drive Th17 differentiation both in vitro and in vivo through enhancing mitochondrial ROS production [12]. Notably, N-acetylcysteine, a specific inhibitor of mitochondrial ROS production, has been reported to significantly inhibit Th17 differentiation [33]. These findings collectively highlight the significant impact of dysregulated redox balance on Th17 differentiation and pathogenicity, underscoring the necessity of proper regulation of ROS to control the development of Th17-related autoimmune diseases. Maintaining the balance between ROS and antioxidant proteins within the cell is critical for preserving the integrity of Th17 cell-mediated immunity. As Nrf2-mediated signaling is a vital antioxidant system for maintaining intracellular redox balance, targeting Nrf2 emerges as a promising therapeutic strategy for Th17-dependent autoimmune diseases.

4. Insights into Nrf2-targeted therapy for Th17-dependent autoimmune diseases

Nrf2 is a stress-sensitive transcription factor that serves as a critical regulator in maintaining cellular redox hemostasis. Upon oxidative stress, Nrf2 translocates from the cytoplasm to the nucleus, where it becomes activated. Once activated, Nrf2 binds to antioxidant response elements (ARE) located in the promoters of target genes, initiating the transcription of ARE-containing cytoprotective antioxidant genes [8]. These antioxidant response genes, downstream of Nrf2, play a pivotal role in maintaining cellular redox balance within a physiological range. This delicate balance ensures proper metabolic processes and is essential for cellular function and viability [7].

Reports have elucidated the impact of Nrf2 on Th17 differentiation and pathogenicity. Deficiency in Nrf2 has been associated with augmented Th17 differentiation and the production of relevant cytokines (e.g., IL-17 and IL-23) both in in vitro experiments and in a murine model of lupus nephritis [34]. In addition, Nrf2-dependent antioxidant gene expression is suppressed in in vitro differentiated Th17 cells, and Nrf2 activation inhibits IL-17a production at both transcript and protein levels in peripheral blood mononuclear cells derived from MS patients. Mechanistically, Nrf2 binds to ARE repressor of Rorc promoter and inhibits Rorc-dependent IL-17 transactivation [35]. Conversely, T cell-specific overexpression of Nrf2 has been shown to promote the development of T regulatory cells [36]. These findings indicate the pivotal role of Nrf2 in modulating immune responses, as it not only prevents the differentiation of pro-inflammatory Th17 cells but also promotes the shift toward an anti-inflammatory T regulatory cell phenotype. Given its regulatory effects on Th17 differentiation and pathogenicity, Nrf2 emerges as a promising therapeutic target for Th17-mediated autoimmune disorders such as MS, psoriasis, and RA.

EAE is a widely used mouse model for MS research. It is induced by the activation of Th17 and Th1 cells, which penetrate the central nervous system, resulting in the degradation of myelin and death of oligodendrocytes [37]. Nrf2-deficient mice exhibit exacerbated EAE outcomes, marked by increased production of pro-inflammatory cytokines in the brain and spinal cord, and enhanced T cell infiltration in the central nervous system [38, 39]. Dimethyl fumarate (DMF) and its metabolite, monomethyl fumarate, demonstrate therapeutic potential by stabilizing Nrf2 and enhancing its transcriptional activity, effectively reducing the severity of EAE [40, 41]. Beyond DMF, other Nrf2 activators, like trifluoroethyl amide derivate of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-TFEA), exhibit promise in EAE management. CDDO-TFEA administration correlates with diminished levels of circulating pro-inflammatory cytokines and increased expression of Nrf2 and its target genes in various immune compartments, including brain, spinal cord, splenocytes, and CNS-infiltrating immune cells [42]. Moreover, compounds such as D3T [43] and TFM-735 [44], both of which activate Nrf2, have demonstrated protective effects during EAE with notable immunomodulatory functions.

RA is characterized by dysregulated Th17-driven inflammation. Studies have demonstrated the impact of Nrf2 on RA pathogenesis. Nrf2 deficiency increased leukocyte infiltration and accelerated the incidence of arthritis in mice [45]. Moreover, Nrf2-deficient mice exhibit more inflammation and oxidative damage in antibody-induced arthritis, a mouse model of RA [46]. Additionally, DMF treatment has been shown to attenuate osteoclastogenesis and bone destruction via induction of Nrf2-mediated transcription of antioxidant genes in a mouse model of LPS-induced arthritis [47]. Furthermore, treatment with S-propargyl-cysteine has demonstrated efficacy in reducing RA-associated inflammation by modulating the Nrf2-ARE signaling pathway [48]. These findings collectively suggest that targeting Nrf2 may provide therapeutic strategy for RA patients.

Th17 is believed to be the key contributor to the inflammatory processes underlying psoriasis, a chronic inflammatory skin disorder. Studies using Nrf2-deficient mice have shown that the mice exhibit increased inflammatory responses to imiquimod-induced psoriasis, as evidenced by increases in ear thickness and elevated mRNA expression of key pro-inflammatory cytokines, such as IL-6, TNFα, IL-17a, and IL-23a, in the ear tissue [49]. Moreover, topical application of Nrf2 siRNA to the ear skin resulted in reduced inflammation in mice with imiquimod-induced psoriasis [50]. DMF has been observed to enhance epidermal barrier function and alleviate the severity of psoriatic lesions [49]. Additionally, treatment with sulforaphane in mice has demonstrated efficacy in ameliorating psoriatic symptoms and protecting against cutaneous inflammation in an Nrf2-dependent manner [51]. The effect of Nrf2 modulation on autoimmune disease is summarized in Table 1. These findings suggest that targeting Nrf2 may serve as a potential therapeutic approach for managing psoriasis.

Disease	Model	Effect of Nrf2	Reference
Mouse lupus nephritis	Nrf2 deficiency	increased Th17 differentiation, increased lupus nephritis	[34]
Mouse EAE	Nrf2 deficiency	exacerbated EAE	[38, 39]
Mouse RA	Nrf2 deficiency	more inflammation and oxidative damage, increased leukocyte infiltration, increased arthritis in mice	[45, 46]
Mouse psoriatic skin inflammation	Nrf2 deficiency	increased inflammation and psoriasis	[49]
Imiquimod-induced psoriasis mouse model	Nrf2 siRNA	reduced inflammation and psoriasis	[50]
PBMC from MS patients	Nrf2 activator	inhibited IL-17a production	[35]
Mouse EAE	Nrf2 activator	reduced EAE	[40, 41, 42, 43, 44]
LPS-induced arthritis in mouse	Nrf2 activator	attenuated osteoclasts	[47]
Imiquimod-induced psoriasis mouse model	Nrf2 activator	alleviated psoriasis severity	[51]

Table 1.

Summary of Nrf2 study in autoimmune disease model.

5. Conclusion

Overall, balancing cellular redox levels for proper metabolic processes is critical for managing Th17-dependent autoimmune diseases. Targeting Nrf2-mediated signaling pathways offers a promising avenue for precision medicine and improving outcomes in such conditions. By modulating Nrf2 activity, it becomes possible to regulate Th17 cell differentiation and function, thereby mitigating autoimmune pathology (Figure 1). This approach holds considerable potential for developing novel therapeutic interventions that can effectively manage Th17-mediated autoimmune diseases.

Figure 1.

Regulation of Th17 and autoimmune disease by Nrf2. Th17 cells are generated from naïve CD4⁺ T lymphocytes upon exposure to a pathogenic environment (e.g., IL-6, TGFβ and IL-23). The differentiated Th17 cells secrete pathogenic cytokines including IL-17, IL-22, and GM-CSF, thereby facilitating the development of autoimmune disease through elevated ROS production. Activation of Nrf2 inhibits ROS and protects against autoimmune disease development.

Abbreviations

ARE	antioxidant response elements
DMF	dimethyl fumarate
EAE	experimental autoimmune encephalomyelitis
ETC	electron transport chain
GLUT	glucose transporter
HIF1α	hypoxia-inducible factor 1α
IL-17	interleukin-17
MS	multiple sclerosis
Nrf2	nuclear factor erythroid 2-related factor 2
OXPHOs	oxidative phosphorylation
PDHK1	pyruvate dehydrogenase kinase 1
PKM2	pyruvate kinase isoform 2
RA	rheumatoid arthritis
RORγt	RAR-related orphan receptor gamma
ROS	reactive oxygen species
TCA	tricarboxylic acid cycle
CDDO-TFEA	trifluoroethyl amide derivate of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid