Blood RNA-sequencing across the continuum of ANA-positive autoimmunity reveals insights into initiating immunopathology.

OBJECTIVE: Mechanisms underpinning clinical evolution to systemic lupus erythematosus (SLE) from preceding antinuclear antibodies (ANA) positivity are poorly understood. This study aimed to understand blood immune cell transcriptional signatures associated with subclinical ANA positivity, and progression or non-progression to SLE. METHODS: Bulk RNA-sequencing of peripheral blood mononuclear cells isolated at baseline from 35 ANA positive (ANA+) subjects with non-diagnostic symptoms was analysed using differential gene expression, weighted gene co-expression network analysis, deconvolution of cell subsets and functional enrichment analyses. ANA+ subjects, including those progressing to classifiable SLE at 12 months (n=15) and those with stable subclinical ANA positivity (n=20), were compared with 15 healthy subjects and 18 patients with SLE. RESULTS: ANA+ subjects demonstrated extensive transcriptomic dysregulation compared with healthy controls with reduced CD4+naïve T-cells and resting NK cells, but higher activated dendritic cells. B-cell lymphopenia was evident in SLE but not ANA+ subjects. Two-thirds of dysregulated genes were common to ANA+ progressors and non-progressors. ANA+ progressors showed elevated modular interferon signature in which constituent genes were inducible by both type I interferon (IFN-I) and type II interferon (IFN-II) in vitro. Baseline downregulation of mitochondrial oxidative phosphorylation complex I components significantly associated with progression to SLE but did not directly correlate with IFN modular activity. Non-progressors demonstrated more diverse cytokine profiles. CONCLUSIONS: ANA positivity, irrespective of clinical trajectory, is profoundly dysregulated and transcriptomically closer to SLE than to healthy immune function. Metabolic derangements and IFN-I activation occur early in the ANA+ preclinical phase and associated with diverging transcriptomic profiles which distinguish subsequent clinical evolution.

Type I interferon blockade with anifrolumab in patients with systemic lupus erythematosus modulates key immunopathological pathways in a gene expression and proteomic analysis of two phase 3 trials.

INTRODUCTION: Anifrolumab is a type I interferon (IFN) receptor 1 (IFNAR1) blocking antibody approved for treating patients with systemic lupus erythematosus (SLE). Here, we investigated the immunomodulatory mechanisms of anifrolumab using longitudinal transcriptomic and proteomic analyses of the 52-week, randomised, phase 3 TULIP-1 and TULIP-2 trials. METHODS: Patients with moderate to severe SLE were enrolled in TULIP-1 and TULIP-2 and received intravenous anifrolumab or placebo alongside standard therapy. Whole-blood expression of 18 017 genes using genome-wide RNA sequencing (RNA-seq) (pooled TULIP; anifrolumab, n=244; placebo, n=258) and 184 plasma proteins using Olink and Simoa panels (TULIP-1; anifrolumab, n=124; placebo, n=132) were analysed. We compared treatment groups via gene set enrichment analysis using MetaBase pathway analysis, blood transcriptome modules, in silico deconvolution of RNA-seq and longitudinal linear mixed effect models for gene counts and protein levels. RESULTS: Compared with placebo, anifrolumab modulated >2000 genes by week 24, with overlapping results at week 52, and 41 proteins by week 52. IFNAR1 blockade with anifrolumab downregulated multiple type I and II IFN-induced gene modules/pathways and type III IFN-λ protein levels, and impacted apoptosis-associated and neutrophil extracellular traps-(NET)osis-associated transcriptional pathways, innate cell activating chemokines and receptors, proinflammatory cytokines and B-cell activating cytokines. In silico deconvolution of RNA-seq data indicated an increase from baseline of mucosal-associated invariant and γδT cells and a decrease of monocytes following anifrolumab treatment. DISCUSSION: Type I IFN blockade with anifrolumab modulated multiple inflammatory pathways downstream of type I IFN signalling, including apoptotic, innate and adaptive mechanisms that play key roles in SLE immunopathogenesis.

A path to Glucocorticoid Stewardship: a critical review of clinical recommendations for the treatment of systemic lupus erythematosus.

Glucocorticoids (GCs) have revolutionized the management of SLE, providing patients with rapid symptomatic relief and preventing flares when maintained at low dosages. However, there are increasing concerns over GC-associated adverse effects and organ damage, which decrease patients' quality of life (QOL) and increase healthcare costs. This highlights the need to balance effective GC use and minimize toxicity in patients with SLE. Herein, we provide an overview of the theoretical considerations and clinical evidence, in addition to the variations and similarities across nine national and eight international recommendations regarding the use of GCs across SLE manifestations and how these compare with real-world usage. In line with this, we propose possible actions toward the goal of GC Stewardship to improve the QOL for patients with lupus while managing the disease burden.

ANA-arthritis: clinical and biomarker characterization of a population for basket trials.

OBJECTIVES: ANA-associated RMDs (ANA-RMDs-SLE, pSS, scleroderma, inflammatory myositis, mixed connective tissue disease (MCTD) and undifferentiated connective tissue disease) are a disease spectrum with overlapping clinical and immunological features. Musculoskeletal inflammation is common and impactful across ANA-RMDs. We evaluated musculoskeletal inflammation (ANA-arthritis) prevalence in a multi-disease ANA-RMD study, assessed its clinical impact across ANA-RMD diagnoses, proposed new basket groupings of patients and evaluated immunological profiles in legacy and new basket contexts. METHODS: An observational study enrolled ANA-RMD patients. Demographic variables, comorbidities, therapies, disease activity instruments (BILAG, SLEDAI, ESSDAI, physician-VAS), patient-reported outcomes (SF36, FACIT-Fatigue, EQ5D, ICECAP-A, WPAI, patient-VAS) and biomarker profile (6 gene expression scores, flow cytometry, autoantibody profile) were analysed. Reclustering utilized Gaussian Mixture Modelling (GMM). Clinical and immune features of new and legacy clusters were compared. RESULTS: Inflammatory MSK symptoms were prevalent across ANA-RMDs, in 213/294 patients. In ANA-arthritis patients, most variables did not differ between diagnoses, excluding EQ5D-5L index and mobility domains (lower in MCTD/pSS, both p< 0.05). Fibromyalgia and osteoarthritis prevalence were similar across diagnoses. Therapy use differed significantly, biologic use being greatest in SLE (p< 0.05).GMM yielded two multi-disease clusters; High-MSK disease activity (n = 89) and Low-MSK disease activity (n = 124). High-MSK disease activity contained all patients with active joint swelling and had significantly higher prednisolone usage, PGA and Sm/RNP/SmRNP/Chromatin positivity, Tetherin-MFI and Interferon Score-A activity; with numerically lower fibromyalgia and osteoarthritis prevalence. CONCLUSION: We define ANA-Arthritis, a more clinically and immunologically homogeneous population than existing RMDs for trials, and a more prevalent population for therapies in the clinic.

Differences in management approaches for lupus nephritis within the UK.

Objectives: Outcomes of therapy for LN are often suboptimal. Guidelines offer varied options for treatment of LN and treatment strategies may differ between clinicians and regions. We aimed to assess variations in the usual practice of UK physicians who treat LN. Methods: We conducted an online survey of simulated LN cases for UK rheumatologists and nephrologists to identify treatment preferences for class IV and class V LN. Results: Of 77 respondents, 48 (62.3%) were rheumatologists and 29 (37.7%) were nephrologists. A total of 37 (48.0%) reported having a joint clinic between nephrologists and rheumatologists, 54 (70.0%) reported having a multidisciplinary team meeting for LN and 26 (33.7%) reported having a specialized lupus nurse. Of the respondents, 58 (75%) reported arranging a renal biopsy before starting the treatment. A total of 20 (69%) of the nephrologists, but only 13 (27%) rheumatologists, reported having a formal departmental protocol for treating patients with LN (P < 0.001). The first-choice treatment of class IV LN in pre-menopausal patients was MMF [41 (53.2%)], followed by CYC [15 (19.6%)], rituximab [RTX; 12 (12.5%)] or a combination of immunosuppressive drugs [9 (11.7%)] with differences between nephrologists' and rheumatologists' choices (P = 0.026). For class V LN, MMF was the preferred initial treatment, irrespective of whether proteinuria was in the nephrotic range or not. RTX was the preferred second-line therapy for non-responders. Conclusion: There was variation in the use of protocols, specialist clinic service provision, biopsies and primary and secondary treatment choices for LN reported by nephrologists and rheumatologists in the UK.

Variants in the DDX6-CXCR5 autoimmune disease risk locus influence the regulatory network in immune cells and salivary gland.

Fine mapping and bioinformatic analysis of the DDX6-CXCR5 genetic risk association in Sjögren's Disease (SjD) and Systemic Lupus Erythematosus (SLE) identified five common SNPs with functional evidence in immune cell types: rs4938573, rs57494551, rs4938572, rs4936443, rs7117261. Functional interrogation of nuclear protein binding affinity, enhancer/promoter regulatory activity, and chromatin-chromatin interactions in immune, salivary gland epithelial, and kidney epithelial cells revealed cell type-specific allelic effects for all five SNPs that expanded regulation beyond effects on DDX6 and CXCR5 expression. Mapping the local chromatin regulatory network revealed several additional genes of interest, including lnc-PHLDB1-1. Collectively, functional characterization implicated the risk alleles of these SNPs as modulators of promoter and/or enhancer activities that regulate cell type-specific expression of DDX6, CXCR5, and lnc-PHLDB1-1, among others. Further, these findings emphasize the importance of exploring the functional significance of SNPs in the context of complex chromatin architecture in disease-relevant cell types and tissues.

Efficacy of anifrolumab across organ domains in patients with moderate-to-severe systemic lupus erythematosus: a post-hoc analysis of pooled data from the TULIP-1 and TULIP-2 trials.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by multisystem involvement. We aimed to evaluate the efficacy of anifrolumab on organ domain-specific SLE disease activity. METHODS: In this post-hoc analysis, data were pooled from the randomised, placebo-controlled, phase 3 TULIP-1 (NCT02446912) and TULIP-2 (NCT02446899) trials of anifrolumab (300 mg intravenously once every 4 weeks for 48 weeks) in patients aged 18-70 years with moderate-to-severe SLE. We evaluated changes from baseline to week 52 in British Isles Lupus Assessment Group 2004 (BILAG-2004) and SLE Disease Activity Index 2000 (SLEDAI-2K) organ domain scores, Cutaneous Lupus Erythematosus Disease Area and Severity Index activity score (CLASI-A), swollen and tender joint counts, haematology, and serology. FINDINGS: Among the 726 patients included, the mean age was 41·8 years (SD 11·9); 674 (93%) were female, 52 (7%) were male, and 479 (66%) were White. 360 patients received anifrolumab 300 mg (180 patients in each trial), and 366 received placebo (184 patients in TULIP-1 and 182 patients in TULIP-2). The most frequently affected organ domains at baseline were musculoskeletal (645 [89%] patients based on BILAG-2004; 684 [94%] with SLEDAI-2K) and mucocutaneous (627 [86%] with BILAG-2004; 699 [96%] based on SLEDAI-2K). At week 52, anifrolumab treatment resulted in greater improvements versus placebo in the musculoskeletal system (176 [56%] of 317 patients vs 143 [44%] of 328 with BILAG-2004; 164 [49%] of 335 vs 141 [40%] of 349 with SLEDAI-2K), the mucocutaneous system (168 [54%] of 315 vs 119 [38%] of 312 with BILAG-2004; 190 [55%] of 348 vs 138 [39%] of 351 SLEDAI-2K), and immunological system (44 [19%] of 237 vs 26 [11%] of 230 with SLEDAI-2K). Less frequently affected domains had varied responses. Among patients with a CLASI-A of 10 or more at baseline, greater proportions of patients receiving anifrolumab than placebo achieved a reduction of 50% or more in CLASI-A at week 52 (49 [46%] of 107 vs 24 [25%] of 94). Among patients with at least six swollen joints, more patients in the anifrolumab group than in the placebo group had a 50% or more reduction from baseline to week 52 in swollen joint count (99 [57%] of 174 vs 92 [46%] of 200), but the difference between groups was not significant for 50% or more reduction in tender joint count. INTERPRETATION: Across the two pivotal phase 3 trials, anifrolumab treatment improved SLE disease activity across multiple organ domains. FUNDING: AstraZeneca.

Lessons for rituximab therapy in patients with rheumatoid arthritis.

B-cell depletion therapy is an effective option for the treatment of rheumatoid arthritis but often does not result in complete B-cell depletion. Complete B-cell depletion after rituximab treatment is associated with clinical response, and this outcome leads to long-term maintenance of therapy. Low pretreatment plasmablast counts, concomitant treatment with disease-modifying antirheumatic drugs, no smoking exposure, the presence of anticitrullinated protein antibodies or rheumatoid factor, and a low interferon signature are all predictive of complete B-cell depletion and clinical response. Half of patients who initially show complete B-cell depletion and clinical response after rituximab treatment eventually lose responsiveness with further infusions. However three-quarters of these patients regain this outcome in their following treatment cycle, suggesting that loss of response is reversible and that patients can still benefit from rituximab retreatment. The efficacy of reduced doses of rituximab is being investigated, but preliminary results suggest that these strategies are best used for maintenance therapy, particularly in patients who suffer adverse events or who are at a high risk of infection. Infusion-related reactions are the most common adverse events associated with rituximab treatment, and monitoring of IgG concentrations is crucial, as low concentrations are correlated with an increased risk of infection.

Type I interferon inhibitor anifrolumab in active systemic lupus erythematosus (TULIP-1): a randomised, controlled, phase 3 trial.

BACKGROUND: Type I interferons are involved in systemic lupus erythematosus (SLE) pathogenesis. In a phase 2 trial, anifrolumab, a human monoclonal antibody to type I interferon receptor subunit 1, suppressed interferon gene signatures and substantially reduced SLE disease activity. Here, we sought to confirm the efficacy of anifrolumab versus placebo in a phase 3 trial of adult patients with SLE and moderate-to-severe disease activity despite standard-of-care treatment. METHODS: TULIP-1 was a double-blind, randomised, controlled, phase 3 trial done at 123 sites in 18 countries. Included patients were aged 18-70 years, with moderate-to-severe SLE, and ongoing stable treatment with either prednisone or equivalent, an antimalarial, azathioprine, mizoribine, mycophenolate mofetil or mycophenolic acid, or methotrexate. Patients were randomly assigned (2:1:2) to receive placebo, anifrolumab 150 mg, or anifrolumab 300 mg intravenously every 4 weeks for 48 weeks. Stable standard-of-care treatment continued except for mandatory attempts at oral corticosteroid tapering for patients receiving prednisone or equivalent of 10 mg/day or more at baseline. The primary outcome was the difference between the proportion of patients who achieved an SLE responder index-4 (SRI-4) response at week 52 with anifrolumab 300 mg versus with placebo. Key secondary outcomes were the difference between the anifrolumab 300 mg group and the placebo group in: proportion of patients in the interferon gene signature test-high subgroup who achieved SRI-4 at week 52; proportion of patients on 10 mg/day or more corticosteroids at baseline who achieved a sustained dose reduction to 7·5 mg/day or less from week 40 to 52; proportion of patients with a cutaneous lupus erythematosus disease area and severity index (CLASI) activity score of 10 or higher at baseline who achieved a 50% or more reduction in CLASI score by week 12; proportion of patients who achieved SRI-4 at week 24; and annualised flare rate through week 52. Other measures of disease activity were also assessed at week 52, including the British Isles Lupus Assessment Group-based composite lupus assessment (BICLA). Safety was also assessed. Efficacy and safety analyses were done in the population of patients who received at least one dose of study drug. This trial was registered at ClinicalTrials.gov (NCT02446912). FINDINGS: Between June 9, 2015, and June 16, 2017, 457 patients were randomly assigned to the anifrolumab 300 mg group (n=180), the anifrolumab 150 mg group (n=93), or the placebo group (n=184). The proportion of patients at week 52 with an SRI-4 response was similar between anifrolumab 300 mg (65 [36%] of 180) and placebo (74 [40%] of 184; difference -4·2 [95% CI -14·2 to 5·8], p=0·41). Similarly, proportions of patients with an SRI-4 response at week 24, and at week 52 in patients in the interferon gene signature test-high subgroup, did not differ between the anifrolumab and placebo groups. In patients with baseline oral corticosteroids of at least 10 mg/day, sustained dose reduction to 7·5 mg/day or less was achieved by 42 (41%) of 103 patients in the anifrolumab 300 mg group and 33 (32%) of 102 patients in the placebo group (difference 8·9 [95% CI -4·1 to 21·9]). In patients with CLASI activity score of at least 10 at baseline, at least 50% reduction by week 12 was achieved by 24 (42%) of 58 patients in the anifrolumab 300 mg group and 14 (25%) of 54 in the placebo group (difference 17·0 [95% CI -0·3 to 34·3]). Annualised flare rates were 0·60 for anifrolumab and 0·72 for placebo (rate ratio 0·83 [95% CI 0·60 to 1·14]). BICLA response was achieved by 67 (37%) of 180 patients receiving anifrolumab 300 mg versus 49 (27%) of 184 receiving placebo (difference 10·1 [95% CI 0·6 to 19·7]). Anifrolumab's safety profile was similar to that observed in phase 2, with similar proportions of patients having a serious adverse event between groups (25 [14%] of 180 for anifrolumab 300 mg, ten [11%] of 93 for anifrolumab 150 mg, and 30 [16%] of 184 for placebo). INTERPRETATION: The primary endpoint was not reached. However, several secondary endpoints, including reduction in oral corticosteroid dose, CLASI responses, and BICLA responses, suggest clinical benefit of anifrolumab compared with placebo. Conclusive evidence for the efficacy of anifrolumab awaits further phase 3 trial data. Despite the inherent limitations of a 1-year phase 3 study, such as incomplete knowledge of applicability to the general population and scarce detection of rare safety signals, in addition to complications from prespecified restricted medication rules, our results suggest that anifrolumab might have the potential to provide a treatment option for patients who have active SLE while receiving standard therapy. FUNDING: AstraZeneca.