Emerging areas for therapeutic discovery in SLE

Abstract

Recent advances in the field of autoimmunity have identified numerous dysfunctional pathways in Systemic Lupus Erythematosus (SLE), including aberrant clearance of nucleic-acid-containing debris and immune complexes, excessive innate immune activation leading to overactive type I IFN signalling, and abnormal B and T cell activation. On the background of genetic polymorphisms that reset thresholds for immune responses, multiple immune cells contribute to inflammatory amplification circuits. Neutrophils activated by immune complexes are a rich source of immunogenic nucleic acids. Identification of new B subsets suggests several mechanisms for induction of autoantibody producing effector cells. Disordered T cell regulation involves both CD4 and CD8 cells. An imbalance in immunometabolism in immune cells amplifies autoimmunity and inflammation. These new advances in understanding of disease pathogenesis provide fertile ground for therapeutic development.

Graphical abstract

INTRODUCTION

Systemic lupus erythematosus (SLE) is a devastating autoimmune disease in which autoantibodies to nuclear antigens cause inflammation and organ damage. Current treatment of SLE relies on broadly immunosuppressive medications with insufficient efficacy and significant toxicities. Despite large efforts in drug development, only one modestly effective biologic, belimumab, is FDA approved for treating active SLE. It is imperative, therefore, that advances in immunologic knowledge drive improvements in the treatment and quality of life of SLE patients. In this review we will summarize newly discovered pathways of immune activation that point to new therapeutic approaches. We will not address the genetic causes of SLE or epigenetic changes in immune cells of SLE patients as these are reviewed elsewhere [1,2]. We also refer readers to a summary of treatments currently in clinical trials [3].

INDUCTION OF SLE BY NUCLEIC ACID ANTIGENS

Following the discovery that dead cells are a continuous source of the major nuclear autoantigens in SLE, advances have been made in defining inflammatory and non-inflammatory types of cell death (reviewed [4]). Safe disposal of dead cell material is a primary function of macrophages that are located in secondary lymphoid tissues and in each organ (Figure 1A). An overload of apoptotic cells beyond the capacity of macrophage disposal, can result in progression to inflammatory forms of cell death that yield nucleic acids in immunogenic form [4,5]. Other sources of excess extracellular and intracellular nucleic acids and the sensors that recognize them are shown in Figures 1B and C.

Figure 1. Innate immune mechanisms in SLE.

1A. An intact pathway for safe recognition and digestion of dead cells by macrophages includes a functional sensing mechanism, upregulation of scavenger receptors and activation of an alternative autophagy pathway called lysosome activated phagocytosis (LAP) that recruits part of the autophagy machinery, including LC3, to single membrane phagosomes and induces the release of regulatory cytokines such as IL-10 and TGFβ. Absence of LAP, but not of canonical autophagy in macrophages results in inefficient clearance of phagosomal material and release of inflammatory cytokines; this is associated with a mild spontaneous form of SLE in mouse models [58].

1B. Extracellular particles containing nucleic acids within immune complexes, microparticles, biofilms and neutrophil NETs are internalized, often via receptor mediated phagocytosis, and encounter endosomal Toll like receptors, TLRs 7, 8 and 9 within phagosomes. These TLRs use the adaptor molecular MyD88 and induce either inflammatory cytokines or Type 1 interferons. Absence of MyD88, IRAK4 and IRF5 all prevent SLE in mouse models. A role for DNAse1L3 in clearance of DNA associated with extracellular microparticles has recently been described [59].

1C. Intracellular nucleic acids engage a different set of sensors. The intracellular DNAse Trex1 clears cytoplasmic DNA; complete deficiency causes an interferonopathy called Aicardi-Goutieres syndrome. Cyclic GMP-AMP synthase (cGAS) catabolizes the formation of the cyclic dinucleotide cGAMP from excess digested cytoplasmic DNA. cGAMP binds to and activates the stimulator of IFN genes (STING), located in the endoplasmic reticulum, resulting in phosphorylation of NFκB and IRFs and the production of inflammatory cytokines. Similarly, cytoplasmic RNA sensors, RIG-I and MDA5 bind to MAVS, a mitochondrial membrane protein that similarly phosphorylates NFκB and IRFs to induce inflammatory cytokines needed for the anti-viral response. An immunogenic role has been defined for oxidized mitochondrial DNA released from activated neutrophils that do not perform mitophagy efficiently. Both TLR9 and STING have been implicated in recognition of neutrophil derived oxidized mitochondrial DNA [15,16]. Mitochondrial DNA released into the cytoplasm during apoptosis may also trigger the activation of cGAS.

Targeting of the innate pathways and sensors involved in induction of inflammatory cytokines and Type I interferons may form the basis for SLE therapy in appropriate patients. Ablation of cGAS in Trex1 deficient mice abrogates SLE, confirming its role as a sensor of cytoplasmic DNA overload [6,7]. It is not clear in how many patients this pathway is engaged; a recent report suggests that ≈15% of SLE patients have increased serum cGAMP levels [8]. Surprisingly, STING deficiency exacerbates SLE in mouse models, reflecting the complex structure of this molecule that has both inflammatory and decoy forms and is also regulated by post-transcriptional modifications [9,10]. MAVS aggregation has been observed in mitochondria of PBMCs from ≈30% of SLE patients, suggesting frequent activation of this pathway [11]. Other targets in the nucleic acid sensing pathway include the endosomal TLRs and their downstream effectors IRAK4 and IRF5 (Figure 1B). Although IRF5 could be a valid target, IRF5 deficient mice develop accelerated atherosclerosis as a result of impaired efferocytosis by macrophages [12]. Finally, patients with high levels of Type 1 interferon may respond to interferon blockade or Jak1 inhibitors. Nevertheless, the disappointing failure of an antibody to the type 1 interferon receptor to reduce disease activity in a recent phase 3 trial, suggests that interferon blockade alone is not sufficient to address the inflammatory burden of active SLE.

The cytokine response induced by nucleic acid sensors resembles that of a chronic viral infection and is accompanied by activation of lymphocytes and, in the appropriate genetic environment, the generation of antibodies to nucleic acids. Nucleic acid-containing immune complexes further activate innate cells that express activating Fc receptors, including plasmacytoid dendritic cells (pDCs) and neutrophils. pDCs are poised to release Type I interferons and their absence in mouse models prevents the initiation of disease [13]. Activated neutrophils release NETs and/or oxidized mitochondrial DNA, both of which amplify the dysregulated immune response. While neutrophil NETs are immunogenic and can deposit in target organs, SLE-prone mice still develop disease in the absence of NET formation [14]. Nevertheless, interest in neutrophils is based on their ability to release immunogenic mitochondrial DNA and on the abnormal neutrophil signature that associates with SLE activity [15–18].

INNATE IMMUNE MECHANISMS ENHANCE AUTOANTIBODY PRODUCTION BY B CELLS

Innate receptors in B cells play a major role in regulating the production of lupus-related autoantibodies. In particular, endosomal TLRs and Type I IFN regulate B cell functions at multiple developmental stages. The disparate roles of TLR7 and TLR9 in B cells have recently been addressed [19,20]. B cell intrinsic TLR7 expression is required for optimal germinal center formation and for the majority of the SLE phenotype observed in TLR7 overexpressing mice [21]. Induction of SLE by excess TLR7 also requires a functional B cell autophagy pathway [22]. IRF5, which is downstream of TLR7, appears to regulate B cell differentiation and plasmablast differentiation [23]. On the other hand, co-engagement of TLR9 and the BCR dampens TLR7 mediated signals and induces apoptosis of naïve B cells unless they are rescued by survival or costimulatory signals [19]. This observation helps explain why TLR9 deficiency exacerbates SLE even though it is required for an anti-DNA response. Because autoreactive B cells recognize and engulf nucleic acid containing material via their BCR and TLR engagement upregulates expression of the BAFF receptor TACI, TLR7 overexpression and/or excess BAFF can preferentially drive the survival and differentiation of such cells [24] (Figure 2).

Figure 2. The intersection of innate and adaptive immunity in regulating autoantibody production in SLE.

Pathways of B cell differentiation and some of the mediators that drive pathway specific differentiation. Upon activation of naïve B cells, TLR9 engagement, CD40 ligation and higher BCR affinity favor the extrafollicular route leading to short term plasma cell differentiation, whereas lower BCR affinity favors the germinal center route. The spontaneous formation of germinal centers is a feature of many mouse models of SLE; recent studies have shown the B cell intrinsic requirement for TLR7, IFNγ receptor and IL6 in their formation [21,60,61]. If B cells enter the germinal center, lower affinity BCR interactions are associated with lower expression of integrins and higher expression of Bcl6; this results in less T cell help and favors the memory cell or recycling GC fate. Long-lived plasma cell differentiation is enhanced when higher BCR affinity facilitates stable interactions with follicular T helper cells in the light zones and is associated with lower expression of Bcl6 and upregulation of BLIMP1 [62]. Surprisingly, in mouse models, excess Type I interferon enhances germinal center reactions but skews plasma cells to a short-lived fate [63]. Orange labels: Stages of B cell differentiation that are influenced by innate immune mediators intrinsic to B cells or interacting with receptors on B cells. Red arrows: Stages of development at which innate signals preferentially induce autoantibodies. Blue arrows: Autoreactive B cells arise routinely but are regulated at every stage of the B cell developmental pathway. Dashed arrows: Potential pathways not fully confirmed. MZ: marginal zone; GC: germinal center; LZ: light zone; DZ: dark zone; ABC: age associated B cell; PB: plasmablast; PC: plasma cell.

TLR8 is expressed in myeloid cells but has been difficult to study in animal models because mouse TLR8 does not recognize RNA. Mice with multiple copies of human TLR8 spontaneously develop multi-organ inflammation with lupus-related autoantibodies and glomerulonephritis [25]. A role for TLR8 has also been proposed in the placental damage caused by anti-phospholipid antibodies [26] and in the induction of T follicular helper cells (TFH) [27].

SOURCES OF AUTOANTIBODIES

Autoantibody production is central to the pathogenesis of SLE. SLE patients have defects in naïve B cell tolerance even during periods of disease quiescence; this is associated with inappropriate responses to TLR9 ligands, decreased cell surface expression of CD19 and CD21 and decreased induction of IL10 [28,29]. What is not known is whether these defective naïve cells are the ones that expand into pathogenic B effector cells. Nor is it known whether the eliciting antigen is always self-antigen or whether other antigen sources are responsible. One potential source of exogenous antigen in the absence of overt infection is the gut, from where bacteria capable of eliciting autoantibody responses may leak through the colon wall [30].

Activated B cells proceed along several different differentiation pathways to become either memory cells or long or short-lived plasma cells (Figure 2). Activated B cells may also differentiate into a subset termed “age associated B cells (ABCs)”. The presence of ABCs correlates with anti-DNA antibodies and hypocomplementemia as well as renal and skin manifestations of SLE [31]. These cells have a unique phenotype, including expression of the myeloid marker CD11c and the transcription factor T-bet and they express a unique transcriptome. ABCs can derive from the extrafollicular pathway, but it is still not clear whether they also routinely arise in a germinal center. They are expanded in mice and humans with SLE, are preferentially autoreactive and are readily differentiated into plasma cells by endosomal TLR ligation together with IFNγ and IL21 produced by activated T cells [32,33]. The cooperation of T-bet with IRF5 in activated ABCs, promotes class switching to IgG2a or IgG2c, maturation into plasma cells and the transcription of genes encoding inflammatory mediators [34].

SLE flares are often associated with the emergence of autoantibody secreting plasmablasts in the peripheral blood. Studies in mice have shown that whether the autoantibody response derives from short or long-lived plasma cells is strain specific and can be skewed by the cytokine milieu. The NZW/BXSB strain has been informative in this regard since the early autoreactive response is germinal center derived but during the late stages of disease, germinal centers disappear and the response becomes extrafollicular [35]. Next generation sequencing studies are beginning to address the origins of plasmablasts in human SLE. Recent studies of patients with SLE flares have shown that the repertoire of these cells is polyclonal, with dsDNA or RNA specific cells accounting for about 1–3% of the repertoire. Importantly, approximately 10–30% of these plasmablasts derive from “naïve activated” B cells that have overlapping characteristics with ABCs, most likely through the extrafollicular route, whereas the rest derive from memory cells [36]. These two pathways of plasmablast differentiation could potentially be sensitive to different therapeutic interventions.

ABNORMALITIES IN SLE T CELLS

SLE T cells are abnormal in several ways. CD4 T cells from active SLE patients have altered signaling and a faster T cell calcium flux than those of healthy individuals due to lysosomal degradation of the CD3ζ signaling component of the TCR complex and its replacement by the FcRγ common chain [37]. This recruits the adaptor molecule Syk rather than ZAP70 and activates the downstream kinase calcium/calmodulin-dependent protein kinase type IV (CaMK4) that enhances production of IL-17 and blocks production of IL-2 [38]. Active SLE patients also have an increase in circulating CXCR5+ (TFH) CD4 T cells [39,40], perhaps skewed to the TFH1 (IFNγ producing) phenotype [41] and they manifest a defect in T regulatory cells (Tregs). The balance between effector T cells and Tregs is regulated by Rho-kinases that can be targeted by specific kinase inhibitors [42]. The administration of low-dose IL2 may be another way to enhance Treg function [43].

A role for CD8 T cells is being re-examined in SLE. Some SLE patients have a phenotype in which a low costimulatory profile of CD4 T cells is associated with an “exhausted” phenotype of CD8 T cells. This phenotype has been associated with a lower incidence of SLE flares and an improved outcome [44]. A recent study has highlighted the role of C1q in suppressing the activation and expansion of CD8 effector T cells in a SLE model, revealing a new function of this molecule in addition to its role in opsonization and non-inflammatory cell clearance [45].

DISTURBANCES OF METABOLIC PATHWAYS

The activation of immune cells is associated with changes in metabolic programs that harness the requisite sources of energy for cell proliferation, protein production, the generation of anti-bacterial and anti-inflammatory mediators and effector subset differentiation. Metabolic reprogramming of T cells in SLE has received much recent attention with the recognition that unlike conventionally activated T cells that have an increase in glycolysis and glutaminolysis, SLE CD4 T cells also manifest an increase of mitochondrial oxidative phosphorylation and abnormalities of lipid metabolism (reviewed [46,47]). Mitochondrial hyperpolarization and oxidative stress in CD4 T cells are associated with an increase in TCR signaling, degradation of CD3ζ, inflammatory cytokine production and poor production of IL2; this phenotype can be reversed by the administration of mTOR inhibitors [48,49] Furthermore, inhibition of the LXRβ receptor can reverse the changes in lipid metabolism and correct lupus-related T cell abnormalities [50]. Most strikingly, combined inhibition of glycolysis and oxidative phosphorylation reverses active disease in mouse models of SLE [51] (Figure 3)

Figure 3. Metabolic pathways are fertile ground for therapeutic interventions in SLE.

Metabolic pathways influence effector function of multiple cell types. Glycolysis is typically associated with inflammatory functions of immune cells whereas oxidative phosphorylation is associated with suppressive or reparative functions. Activated CD4 T cells in SLE have enhanced glycolysis and oxidative phosphorylation. Metformin inhibits mitochondrial complex 1, thereby inhibiting oxidative phosphorylation, but also acts via the mTOR pathway to increase fatty acid oxidation and glycolysis. The latter is inhibited by 2 deoxy-glucose (2DG), explaining why the combination of metformin and 2DG can reverse active SLE in mouse models, whereas neither drug can do this alone [51].

The function of other immune cells is also highly influenced by their metabolic state. Macrophage differentiation depends on metabolic switches; use of glutamine and activation of the arginosuccinate shunt promotes the function of inflammatory macrophages, whereas alternatively activated macrophages use fatty acid oxidation and have an intact Krebs cycle [52]. Activated B cells are predominantly glycolytic; this pathway is enhanced by high levels of BAFF [53]. Interestingly, long-lived plasma cells have a higher respiratory capacity and take up more glucose than their short-lived counterparts; this is converted to pyruvate which is required for their long-term survival (reviewed [54]). Potential approaches for targeting specific immune functions in human SLE using metabolic inhibitors have been recently reviewed [46,49].

ORGAN DAMAGE

New application of kinase inhibitors or anti-cytokine antibodies may add to the anti-inflammatory armamentarium available for the treatment of SLE [3]. It is increasingly appreciated, however, that organ damage in SLE patients involves mechanisms that may not respond well to standard immunosuppressive drugs. Neurologic damage may occur via antibody-mediated cytotoxicity of neurons by antibodies that cross a damaged blood brain barrier and/or by synaptic pruning of neurons by activated microglia. Placental damage induced by anti-phospholipid antibodies is due to trophoblast activation and complement-mediated inflammation. Premature myocardial infarctions and strokes are due to accelerated atherosclerosis that is associated with loss of protective function of circulating HDL, endothelial dysfunction and the presence of low density granulocytes. Progression of SLE nephritis involves progressive interstitial fibrosis and metabolic dysfunction of tubular cells leading to tubular atrophy. Readers are referred to recent reviews addressing these areas [26,55–57].

CONCLUSION: APPLICATION OF NEW KNOWLEDGE TO IMPROVED PATIENT CARE

Inappropriate patient selection for clinical trials not only exposes patients to potential toxicities but poses the risk for failure if the responding patient subset is diluted with non-responders. The current challenge in SLE is to find the underlying immune mechanisms that are perturbed in each patient so that one can ask whether that particular pathway can be normalized and, if so, at which disease stage this approach should be applied. Advances in therapy will require both a better understanding of disease pathogenesis and new approaches to therapy (Box 1). Finally, it is important to recognize that wide disparities exist in patient access to optimal medical care for SLE. Development of new therapies must therefore be accompanied by changes in health policy that bring the needed improvements in care to all SLE patients.

Box 1: Some current areas for investigation in SLE.

• Disease pathogenesis

  • Which innate nucleic acid sensors are crucial in inducing or protecting from SLE?
  • What are the origins of the plasma cells that produce autoantibodies and what is their relationship to protective antibody responses?
  • What are the metabolic changes associated with autoimmune T and B cells?
  • What other pathways e.g. microbiome, neurologic inputs, contribute to the dysregulated immune response?
  • What are the mechanisms for tissue damage in each organ?
• Stratifying patients for clinical trials

  • Can we identify the source of immunogenic nucleic acids and the relevant innate sensors in individual patients?
  • Can patients with SLE be stratified based on their genetic, epigenetic or modular profiles [64,65]?
  • Are there biomarkers that distinguish SLE patients based on either immune mechanisms for tolerance loss or mechanisms of target organ damage?
• Have we exhausted therapies that target adaptive immune responses?

  • Is there a way to tolerize to lupus-related autoantigens without compromising protective or anti-tumor immune responses?
  • Will strategies for induction of T regulatory cells prevent or treat SLE?
  • Is it possible to induce the CD8 T cell exhaustion phenotype?
  • Is it possible to target metabolic pathways without compromising protective immunity or the function of vital organs?
• Targeting disease stages

  • What is the role of animal models of disease in testing immune interventions?
  • Can we enhance clearance of immunogenic nucleic acid containing material to prevent disease initiation or flares?
  • Are there current or experimental therapies that will synergize and how should these be tested [66]?
  • Can we identify and safely treat the preclinical phase of disease?
  • Is it more effective and safer to target quiescent vs. active disease?
  • Can we design therapies specific for injury mechanisms in each organ?
  • Is it possible to arrest or reverse tissue fibrosis?

HIGHLIGHTS.

• Nucleic acid sensors form a bridge between innate and adaptive immunity to regulate tolerance in SLE

• Autoantibody-forming cells in SLE are clonally diverse and derive from both naïve and memory cells

• Metabolic dysfunction in SLE CD4 T cells causes imbalance of CD4 effector and regulatory cells

• Varying organ-specific mechanisms for damage may require additional non-immune interventions