A review of advances in the understanding of lupus nephritis pathogenesis as a basis for emerging therapies

Rituximab

CD20 is a specific B cell surface antigen that promotes differentiation and activation. It is expressed on immature, mature, and activated B cells and is absent on hematopoietic stem cells, pro-B cells, and plasma cells^17,18. Rituximab is a type I chimeric IgG1 mouse/human monoclonal antibody directed against CD20, and it depletes B cells, thus diminishing their differentiation into plasma cells, and therefore decreases autoantibody production¹⁹. Rituximab was originally approved for the treatment of relapsed or refractory, low-grade or follicular, CD20⁺ B cell non-Hodgkin lymphoma, and its use has been extended to various autoimmune diseases such as rheumatoid arthritis, ANCA-associated vasculitis, and primary immune thrombocytopenic purpura²⁰. Crosslinking of CD20 molecules by rituximab induces the redistribution of CD20 into membrane microdomains known as lipid rafts and the formation of antigen–antibody complexes, leading to CD20⁺ B cell apoptosis through three mechanisms, namely complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and, to a lesser extent, direct signaling through CD20. Rituximab eliminates peripheral B cells without suppressing their regeneration from B cell precursors, and, by sparing plasma cells, has no immediate effect on Ig level. In preclinical studies, treatment of New Zealand black and white, first generation (NZB/W F1) mice with anti-CD20 antibodies before the onset of proteinuria delayed disease onset, whereas treatment after onset of nephritis reduced disease progression, suggesting that B cells are critical to both the initiation and the maintenance of disease²¹. There is also a subset-associated hierarchy of B cell sensitivity to depletion by rituximab, with peripheral blood B cells being the most sensitive, and B cells in the spleen, lymph node, or bone marrow being less sensitive, which could be related to relatively low tissue penetration by the antibody. Despite its primary action on B cells, the B cell depletion that resulted from treatment with rituximab was associated with a reduction in T cell memory and activation. Some NZB/W F1 mice appeared to be resistant to the B cell-depleting action of rituximab, which was attributed to a rapid clearance of circulating anti-CD20 antibodies and/or increased BAFF secretion²¹.

Data from the LUNAR trial showed that rituximab, when added to standard induction immunosuppressive regimens, did not increase the rate of renal response, despite leading to more pronounced improvements in serological parameters and proteinuria²². Proposed explanations for this apparent paradox include the efficacy of background immunosuppression, suboptimal depletion of tissue-infiltrating B cells and plasma cells, increased BAFF secretion, and inadequate sensitivity of clinical efficacy endpoints. Lack of treatment efficacy could also be related to monoclonal antibody metabolism, reduced antibody binding due to FcγRIII polymorphism, complement depletion, increased expression of complement inhibitory molecules such as CD55 and CD59, abnormal lipid raft composition, or limited access to the parenchyma of solid organs^23,24. In this regard, B cells are present in the tubulo-interstitium and contribute to local inflammation and kidney injury²⁵. It is currently unknown to what degree rituximab can directly deplete B cells in the kidney. Another mechanism for rituximab resistance may involve the upregulation of CD46, a key surface protein that inhibits complement activation. Serum CD46 level, possibly a product of proteolytic cleavage from apoptotic cell membranes, is increased in SLE patients with active disease compared to patients with inactive disease^26,27. Whether combination therapy targeting both CD46 and CD20 may increase efficacy remains to be investigated. Despite the negative results in pivotal trials, rituximab is commonly used in the management of SLE patients, including patients with LN, in view of the favorable real-world clinical experience in severe renal or extra-renal manifestations and its relatively low toxicity, especially in patients who have an inadequate response to or who cannot tolerate standard therapies. Results from post-hoc analysis of the LUNAR trial also suggested that it may be particularly useful in high-risk patient groups such as Afro-Americans^28–32.

Obinutuzumab

Obinutuzumab is a glycoengineered, type II, humanized anti-CD20 antibody developed to treat lymphoproliferative disorders and to alleviate several mechanisms which could lead to treatment resistance. Obinutuzumab has no fucosylated sugars on the Fc portion and demonstrates enhanced binding affinity to the FcγRIII receptor on immune effector cells²⁴. Obinutuzumab is more effective than rituximab in inducing direct B cell death and antibody-dependent cellular cytotoxicity and phagocytosis. Unlike rituximab, it does not relocalize CD20 to lipid rafts and its effect on B cell depletion is not influenced by complement protein exhaustion or complement-inhibiting factors²⁴. Pre-clinical data showed the superior efficacy of obinutuzumab in B cell depletion compared with rituximab²⁴. Recent results from the phase II NOBILITY study (NCT02550652) showed that obinutuzumab, when added to background immunosuppression comprising corticosteroids and mycophenolate, increased the rates of achieving primary and secondary efficacy endpoints, possibly related to a rapid and complete depletion of peripheral, memory, and naïve B cell subsets and plasmablasts^33,34.

Belimumab and agents that target the BAFF/APRIL pathway

BAFF and a proliferation-inducing ligand (APRIL) are transmembrane proteins of the TNF family synthesized by myeloid cells. They can be proteolytically cleaved from the cell and exist in soluble form. BAFF and APRIL bind to receptors such as transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA). BAFF and APRIL are required for B cell survival from their initial development to terminal differentiation. BAFF promotes the survival and maturation of transitional B cells into mature B cells and supports B cell proliferation, plasma cell survival, and class-switch recombination. APRIL also promotes cell survival and class-switch recombination and plays an important role in T cell-independent responses¹³. Inhibition of B cell survival factors represents an alternative approach to inhibit B cell and humoral immune responses. Belimumab is a human IgG1λ antibody that blocks the bioactivity of soluble BAFF, thus inhibiting B cell survival and differentiation. Results from two randomized controlled trials (RCTs), BLISS-52 and BLISS-76, demonstrated the efficacy of belimumab in active SLE patients, but patients with moderate or severe LN or neuropsychiatric complications were excluded from these trials^35,36. Post-hoc analysis of the pooled data from these trials suggested an efficacy of belimumab treatment on renal manifestations and a decreased incidence of renal flare in patients who received belimumab³⁷. Whether the latter could be explained by sustained inhibition of memory B cells and plasma cells remains speculative³⁸. A phase III clinical trial which investigated the efficacy of belimumab in LN (BLISS-LN; NCT01639339) was completed recently, and the results showed that a higher number of patients met its primary and secondary endpoints when treated with belimumab plus standard induction therapy compared to placebo and standard induction therapy³⁹.

While data on belimumab in SLE and LN appear to be encouraging, other anti-BAFF therapies have failed to demonstrate efficacy in clinical trials. Results from the ILLUMINATE-1 and -2 trials showed that treatment with tabalumab, a monoclonal antibody against both soluble and membrane-bound BAFF, did not meet the primary efficacy endpoint of SLE responder index (SRI)-5 in patients with active SLE but no renal involvement^40,41, and pooled data from these two RCTs did not suggest beneficial effects of tabalumab on the renal outcomes⁴². Atacicept is a human recombinant fusion protein of TACI and the Fc portion of IgG1. The APRIL-LN trial (NCT00573157), which aimed to investigate the role of atacicept in the treatment of active LN, was terminated early because of severe infective complications associated with low serum Ig level in the first few treated patients⁴³. Telitacicept, also known as RC18, is a novel recombinant TACI-Fc fusion protein that targets and inhibits BLyS (aka BAFF) and APRIL and suppresses the development and survival of plasma cells and mature B cells. A recent phase IIb study (NCT02885610) demonstrated that telitacicept in combination with standard therapy met its primary endpoint in active SLE patients without nephritis⁴⁴. Sequential rituximab-belimumab treatment in patients with active LN is a therapeutic approach currently being investigated (CALIBRATE; NCT02260934), aiming to ensure adequate B cell suppression by first depletion and then preventing their repopulation using belimumab⁴⁵. Interim data from the CALIBRATE study showed that despite B cell depletion and delayed circulating B cell reconstitution, sequential rituximab-belimumab treatment did not improve clinical outcome⁴⁵. Results from the SynBioSe study (NCT02284984) demonstrated that combination treatment with rituximab and belimumab in SLE patients with severe and refractory disease improved clinical outcome, and this was associated with decreased production of neutrophil extracellular traps (NETs)⁴⁶. A phase II clinical trial (SynBioSe-2; NCT03747159) will investigate the efficacy of belimumab followed by rituximab in LN patients. A phase III clinical trial (BLISS-BELIEVE; NCT03312907) investigating the efficacy and safety of belimumab administered in combination with rituximab in patients with active SLE is ongoing⁴⁷. Another ongoing study (phase II; NCT04058028) is investigating the use of BAFF/inducible T-cell co-stimulator ligand (ICOSL) bispecific antibody in active SLE patients without recent cerebral or renal involvement.

Other therapies targeting B cells or plasma cells

B cell depletion can also be achieved by monoclonal antibodies binding to other B cell surface molecules such as CD19 (XmAb5871). In a recent phase II clinical trial (NCT02725515), XmAb5871 treatment of non-organ-threatening SLE patients did not meet the primary endpoint of “no loss of improvement (LOI)” but achieved the secondary endpoint of “time-to-LOI and safety”⁴⁸. Emerging evidence suggests that proteasome inhibitors such as bortezomib and ixazomib (an oral proteasome inhibitor with fewer neurological complications, such as a lower risk of peripheral neuropathy) can target plasma cells through binding to 26S proteasome and inhibiting its chymotrypsin-like activity and inactivating the nuclear factor kappa-light chain enhancer of activated B cell process. Preliminary clinical experience showed the efficacy of bortezomib in active LN patients who were refractory to conventional induction therapies^49,50, although the high rates of treatment discontinuation due to poor drug tolerability remains a concern. Phase II clinical studies are currently underway to investigate the use of bortezomib in SLE patients (NCT02102594) and ixazomib in LN patients (NCT02176486). The MISSION study (NCT03393013) is a phase Ib/II trial that investigates the safety and tolerability of KZR-616, a specific immunoproteasome inhibitor, in patients with active SLE with or without LN (phase Ib) or patients with active proliferative LN (phase II). Results from the phase Ib study showed that KZR-616 at a dose of 45 mg SC was safe and well tolerated⁵¹, and the phase II clinical trial is still ongoing.

Aiolos and Ikaros are members of the Ikaros family of hemopoietic-specific zinc-finger transcription factors that play key roles in the regulation of lymphocyte development and differentiation^52,53. Ikaros is expressed in pluripotent hemopoietic stem cells and regulates both lymphoid and myeloid cell development. Through its ability to regulate the development of plasmacytoid dendritic cells, Ikaros can also regulate key inflammatory pathways such as signal transducer and activator of transcription (STAT) 4 and IFN-α pathways⁵⁴. Aiolos contributes to B cell development and facilitates the generation of long-lived high-affinity bone marrow plasma cells. Aiolos is weakly expressed in pro-B cells and is increased in pre-B cells and mature peripheral B cells⁵⁵. Studies have shown that Aiolos expression is increased in peripheral blood mononuclear cells (PBMCs) from SLE patients compared to healthy subjects^56,57. CC-220 (iberdomide) is a modulator of the cullin ring ligase 4-cereblon (CRL4^CRBN) E3 ubiquitin ligase complex, which induces ubiquitination of the CRBN substrates Aiolos and Ikaros, resulting in their proteasomal degradation. In vitro studies demonstrated that the addition of iberdomide to B cells or PBMCs from SLE patients or healthy subjects, respectively, reduced Aiolos and Ikaros expression and inhibited BAFF-induced B cell differentiation and the production of anti-dsDNA antibodies and anti-phospholipid antibodies^56,57. A phase IIa clinical study (NCT02185040), which investigated the tolerability and effect of iberdomide on skin, joint, and serological manifestations in SLE patients, reported that iberdomide significantly reduced B cell subset populations and plasmacytoid dendritic cells and improved arthritis and cutaneous lupus erythematosus disease area and severity index (CLASI) activity score, an assessment score for cutaneous involvement^58,59. A phase IIb clinical study (NTC03161483) to investigate the efficacy and safety of iberdomide in a larger cohort of patients with active SLE is ongoing.

Mammalian or mechanistic target of rapamycin

The mammalian or mechanistic target of rapamycin (mTOR) is an evolutionarily conserved serine-threonine kinase that plays a key role in the regulation of cell proliferation, metabolism, and survival. Increased mTOR activation is observed in patients and mice with LN⁹⁵. Sirolimus is a naturally occurring macrolide antibiotic produced by Streptomyces hygroscopicus and is a specific inhibitor of the mTOR pathway. It is a potent inhibitor of B and T cell proliferation. In pre-nephritic NZB/W F1 mice, treatment with sirolimus suppressed lymphoproliferation, reduced monocyte chemoattractant protein-1 (MCP-1) expression in the kidney, and delayed phenotypic manifestation of disease⁹⁶. Sirolimus treatment given after the onset of nephritis in NZB/W F1 mice was effective in decreasing anti-dsDNA level, improving kidney histopathology, and prolonging survival^97–99. There is also emerging evidence that the anti-proliferative property of mTOR inhibitors extends beyond lymphocytes to non-immune cells. We recently demonstrated that sirolimus decreased mesangial cell proliferation and their binding by anti-dsDNA antibodies and suppressed fibrotic responses in mesangial cells when cells were exposed to anti-dsDNA antibodies or transforming growth factor (TGF)-β1, and the effect was mediated through down-regulation of mTOR and extracellular signal-regulated kinase phosphorylation⁹⁵. In NZB/W F1 mice with active nephritis, sirolimus was as effective as mycophenolate in attenuating kidney inflammation and fibrosis⁹⁵. A recent single-arm phase I/II study (NCT00779194) reported that sirolimus treatment in patients with active SLE but without nephritis or life-threatening lupus complications resulted in a decrease in disease activity score and a T cell profile indicating a less pro-inflammatory phenotype¹⁰⁰. We have also reported our experience of the use of sirolimus in LN patients, suggesting that this group of drugs may have a role in LN management, especially in patients who do not tolerate other treatments or who could derive benefit from the unique properties of these drugs, such as a lower rate of malignancies^101,102.

Janus kinase-1, tyrosine kinase-2, and Bruton’s tyrosine kinase signaling pathways

The success of Janus kinase (JAK) inhibition in murine lupus has prompted clinical trials on JAK inhibitors in human SLE. One phase II RCT (NCT02708095) demonstrated the efficacy of baricitinib (a JAK1/2 inhibitor) in alleviating arthritis in SLE patients¹⁰³. A phase IIb study (PF-06700841) will investigate a selective JAK1 inhibitor in moderate-to-severe active SLE patients without renal or cerebral involvement who show an inadequate response to standard therapies. Inhibition of JAK1 should be used with caution, as a phase II clinical trial on solcitinib (a selective JAK1 inhibitor) (NCT01777256) was discontinued because of severe drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome and hepatic function abnormalities^104,105. Tyrosine kinase 2 (TYK2) is a member of the JAK family and functions downstream of IL-12, IL-23, and type I IFN signaling. A phase II RCT (PAISLEY; NCT03252587) will evaluate the safety and efficacy of BMS-986165, a specific TYK2 inhibitor, in active SLE patients with joint and/or skin involvement¹⁰⁶, and patients who successfully complete the protocol-required treatment period will be recruited to a subsequent phase II trial that will investigate the long-term safety and efficacy (NCT03920267). Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase expressed in B cells and myeloid cells, and it plays an important role in B cell development, survival, and activation and mediates TLR signaling in macrophages. Inhibition of BTK in lupus-prone mice after disease onset reduced total splenic B cell number and suppressed B cell activation, accompanied by decreased proteinuria and improvement of serological parameters¹⁰⁷. Treatment of moderate-to-severe active SLE patients without cerebral or renal involvement with the BTK inhibitor fenebrutinib reduced CD19⁺ B cells, anti-dsDNA antibody titer, and BTK-dependent signature in plasmablasts, but the primary clinical efficacy endpoint (SRI-4) was not met¹⁰⁸.

Cytokines

The importance of type I IFN in SLE and LN is well established. Anifrolumab is a monoclonal antibody against type I IFN receptor subunit 1, and the results from a phase IIb clinical trial (MUSE) showed that anifrolumab treatment significantly suppressed type I IFN gene signatures and lupus disease activity¹⁰⁹. The results from two subsequent phase III RCTs (TULIP-1 and -2) also showed the clinical benefits of anifrolumab in moderate-to-severe active SLE patients. Although the primary endpoint (SRI-4) was not achieved in TULIP-1, anifrolumab treatment could facilitate corticosteroid reduction and improve disease activity scores such as the CLASI and BICLA scores¹¹⁰. The results from TULIP-2 corroborated with TULIP-1: anifrolumab met its primary endpoint (BICLA scores at 52 weeks) in active SLE patients¹¹¹. Consistent with the scientific rationale, the clinical benefit of anifrolumab appears to be more evident in patients with high type I IFN signatures¹¹¹.

Given the pathogenic significance of the IL-17/IL-23 axis in SLE and LN, inhibition of these cytokines may potentially ameliorate SLE and LN. Secukinumab is a fully humanized IgG1κ monoclonal anti-IL-17A antibody and is an approved treatment for active psoriatic arthritis and ankylosing spondylitis, and a phase II clinical trial is underway to investigate its role in patients with refractory LN (NCT04181762). Guselkumab is an anti-IL-23 monoclonal antibody licensed for the treatment of plaque psoriasis, and its use in LN will be explored in a forthcoming phase II study. IL-2 regulates CD4⁺ T cell development and survival, and defective IL-2 production leads to dysregulation in the immune system in SLE patients^112,113. Previous clinical trials demonstrated that treatment of SLE patients with low-dose IL-2 promoted regulatory T (Treg) cells and inhibited T_H17 cells and was associated with the induction of remission^114,115. One recent RCT showed that low-dose IL-2 treatment (1 million IU subcutaneously every other day for 2 weeks) in active SLE patients did not meet the primary efficacy endpoint of SRI-4 at 12 weeks but achieved higher remission rates than placebo in patients with LN and was well tolerated¹¹⁶. The low-dose IL-2 group also showed expansion of Treg and natural killer cells¹¹⁶.

Complement cascade

The complement cascade plays an essential role in innate immunity and comprises more than 30 circulating proteins. The complement system can be activated through one of three initiation pathways, namely the classical pathway, mannose-binding lectin pathway, and the alternative pathway¹¹⁷. The three pathways converge at C3, resulting in the cleavage of C3 to the activated components C3a and C3b. Dysregulation of complement activation is observed in patients and mice with LN. In LN, deposition of C3-containing immune complexes in the glomeruli initiates tissue injury, whereas deficiencies of some of the components of the classical complement pathway, such as C1q and C4, are associated with an increase in the incidence of lupus in humans and also lupus-like disease in C1q or C4 knockout mice¹¹⁸. APL-2 (pegcetacoplan) is a small, synthetic cyclic peptide that binds and inhibits C3 and C3b and interferes with the formation of C3 and C5 convertases as well as inhibits the activity of pre-formed C3 and C5 convertases¹¹⁹. A phase II clinical trial investigated the safety and efficacy of daily APL-2 subcutaneous infusion, administered for 16 weeks with a 6-month safety follow-up, in patients with various glomerulopathies including LN (DISCOVERY; NCT03453619). Preliminary experience from two LN patients recruited into this trial suggests that APL-2 is well-tolerated and treatment was associated with a reduction of proteinuria¹²⁰. Yet the pharmaceutical company has decided to stop the development program for APL-2 in LN after completion of the phase II trial and prefers to focus on other diseases¹²¹.

Treatment of pre-nephritic NZB/W F1 mice with a monoclonal antibody that inhibits C5 cleavage to the potent pro-inflammatory and pro-thrombotic molecules C5a and C5b-9 delayed the development of proteinuria, decreased anti-dsDNA antibody level, improved kidney histopathology, and prolonged survival¹²². Eculizumab is a recombinant fully humanized hybrid IgG2/IgG4 monoclonal antibody that directly binds human complement component C5 and prevents its cleavage to C5a and C5b-9, thereby blocking terminal complement activation while preserving the functions of early complement components. The success of inhibiting C5 cleavage in murine lupus has prompted a single-dose placebo-controlled phase I study to investigate the safety, pharmacodynamics, and pharmacokinetics of eculizumab in 24 SLE patients. While eculizumab at 4 or 8 mg/kg was well tolerated and complement level was reduced to baseline level 2 weeks after commencement of the study, clinical parameters were similar to the placebo group. Relatively low baseline disease activity was proposed as a possible reason for the lack of apparent clinical efficacy associated with eculizumab treatment^123,124. A multicenter phase II clinical trial was designed to investigate the effect of eculizumab in patients with proliferative LN, but the study encountered logistic delays and was terminated¹²⁵. Eculizumab has been approved for the treatment of atypical hemolytic uremic syndrome (aHUS) to inhibit C5-mediated thrombotic microangiopathy¹²⁶. Eculizumab has been reported to be effective in the treatment of a patient with catastrophic antiphospholipid antibody syndrome¹²⁷ and also in a patient with refractory LN¹²⁸. It has been proposed that eculizumab may be beneficial in the treatment of LN patients who have a thrombotic component analogous to aHUS, evidence of C5-driven glomerular inflammation, or antiphospholipid syndrome, although further studies are warranted. It is noteworthy that inhibition of complement activation at the level of C5 is associated with Neisseria infection and prophylactic measures may be required¹¹⁸.

Dysregulation of the classical complement pathway contributes to the pathogenesis of SLE, and there is emerging evidence to show that the alternative pathway also plays a significant role. The alternative pathway is triggered by the activation of factor B, a trypsin-like serine protease that is the proteolytically active component of C3 and C5 convertases. Factor B serves as an acute-phase protein and also as a B cell growth factor. Data from 222 Chinese patients with LN showed reduced plasma C1q and C3 levels and increased levels of factor B, C3a, C5a, and soluble C5b-9¹²⁹. Plasma factor B level correlated with that of C5a and soluble C5b-9, and factor B colocalized with C3b and C5b-9 deposits in the glomeruli¹²⁹. MRL/lpr mice deficient in factor B showed reduced proteinuria, fewer active serological parameters, and improved kidney histology and vasculitis features compared to wild-type and heterozygous mice¹³⁰. The association between factor B deficiency and improved disease manifestation may be attributed to decreased C3 activation and/or B cell development. These data suggest that factor B may present a novel therapeutic target in LN¹³⁰. A highly potent, reversible, and selective small molecule factor B inhibitor (LNP023) is available^131,132. Oral administration of LNP023 to mice with KRN-induced arthritis reduced overall clinical score and inhibited complement activation and infiltration of immune cells in the joints¹³¹. In a rodent model of membranous nephropathy, LNP023 administration before disease onset suppressed the development of proteinuria, while treatment after disease onset delayed progression and attenuated glomerulopathy¹³¹. Phase II clinical studies will investigate the safety, efficacy, tolerability, pharmacokinetics, and pharmacodynamics of LNP023 in patients with paroxysmal nocturnal hemoglobinuria (NCT03439839 and NCT03896152) and IgA nephropathy (NCT03373461).

OMS721 (narsoplimab) is a human monoclonal antibody that targets mannan-binding lectin serine protease 2 (MASP-2), the effector enzyme of the lectin pathway. A phase II study is ongoing to investigate the safety of OMS721 in patients with glomerular diseases including active LN and will look into its potential steroid-sparing effect (NCT02682407). Preliminary results appear encouraging, with treated patients showing a significant reduction of proteinuria (mean reduction 69%) and the feasibility for steroid tapering¹³³. A phase III study will investigate the effect of OMS721 in LN patients¹³⁴.

Neutrophil extracellular traps

Neutrophils play a critical role in the innate immune response, and they also regulate the adaptive immune response¹³⁵. Neutrophil cell death, termed NETosis, results in the formation of NETs, which are composed of nuclear components such as DNA and histones decorated with proteins from primary, secondary, and tertiary granules such as myeloperoxidase, neutrophil elastase, pentraxin 3, and matrix metalloproteinase 9. Under physiological conditions, neutrophils release NETs as a defense mechanism to trap and kill microorganisms¹³⁵. NET formation can also be induced by sterile stimuli such as pro-inflammatory cytokines, immune complexes, or autoantibodies^135,136. In SLE, NET formation is dysregulated and can be induced by ribonucleoprotein antibodies through FcγRIIB (also known as CD32) binding, by the antimicrobial peptide LL37, and by IFN-α produced by plasmacytoid dendritic cells¹³⁷. SLE-induced NETs are believed to be highly immunogenic and contain oxidized mitochondrial DNA, HMGB1, LL37, and immunostimulatory molecules^137,138. The degradation and clearance of NETs in SLE patients are dependent on DNase 1, but DNase 1 activity is impaired in lupus because of the presence of either DNase 1 inhibitors or anti-NET antibodies that inhibit the access of DNase 1 to the NETs, and components of the increased NETs then trigger downstream tissue injury and inflammation^135,139,140. The inability to degrade NETs is associated with a higher propensity for LN¹⁴⁰. In lupus-prone mice, inhibition of NET formation using Cl-amidine (a chemical inhibitor of peptidylarginine deiminase-4)¹⁴¹, DNase 1¹⁴², or FR139317 (a specific endothelin-A receptor antagonist)¹⁴³ resulted in delayed onset of disease, decreased proteinuria, and reduced deposition of immune complexes in the glomeruli as well as down-regulation of IFN-signature inflammatory responses in the bone marrow and kidney^141–143, suggesting that targeting NETs may be beneficial to lupus patients. Tofacitinib, a JAK/STAT inhibitor, has been shown to reduce NET formation in lupus-prone mice, and this was associated with improvements in proteinuria and serological parameters¹⁴⁴. An ongoing phase I study is investigating the safety of tofacitinib in patients with SLE (NCT02535689). Combination therapy with rituximab and belimumab has also been shown to decrease immune complex-induced NET formation in SLE patients⁴⁶. Metformin has been shown to reduce NET formation in neutrophils stimulated with phorbol 12-myristate 13-acetate and decreased IFN-α secretion in plasmacytoid dendritic cells stimulated with mitochondrial DNA¹⁴⁵. Data from a study in Chinese patients (ChiCTR-TRC-12002419) showed that metformin added to conventional treatment for 12 months in SLE patients with mild-to-moderate disease resulted in a decrease in SLE disease activity index (SLEDAI) score by over 50%¹⁴⁵. Mitochondrial DNA is deposited in NETs in renal biopsy specimens from LN patients, and mitochondrial DNA has been shown to induce higher levels of IFN-α in plasmacytoid dendritic cells compared with anti-dsDNA antibodies. In the MUSE trial, SLE patients with a high type I IFN signature showed higher plasma levels of neutrophil granule constituents such as myeloperoxidase, human neutrophil elastase, and citrullinated histone H3 levels, and treatment with anifrolumab for 1 year resulted in a significant decrease in circulating neutrophil NET complexes compared to the placebo group¹⁴⁶.