Neutrophils isolated from systemic lupus erythematosus patients exhibit a distinct functional phenotype.

Neutrophil dysregulation, particularly of a low-density subset, is associated with systemic lupus erythematosus (SLE); however, the exact role of normal-density neutrophils in SLE remains unknown. This study compares activation and functional phenotypes of neutrophils from SLE patients and healthy controls to determine potential contributions to SLE pathogenesis. Surface activation markers and release of neutrophil extracellular traps (NETs), granule proteins, and cytokines/chemokines were measured in resting and stimulated neutrophils from SLE patients (n=19) and healthy controls (n=10). Select miRNA and mRNA involved in neutrophil development and function were also measured. Resting SLE neutrophils exhibited fewer activation markers compared to control neutrophils, and activation markers were associated with different plasma cytokines/chemokines in SLE patients compared to healthy controls. However, activation markers increased similarly in SLE and control neutrophils following stimulation with a TLR7/8 agonist, neutrophil growth factors, and bacterial mimic. At the resting state, SLE neutrophils produced significantly more CXCL10 (IP-10), with trends toward other increased cytokines/chemokines. Following stimulation, SLE neutrophils produced fewer NETs and proinflammatory cytokines compared to control neutrophils but more MMP-8. In addition, SLE neutrophils expressed less miR130a, miR132, miR27a, and miR223. In conclusion, SLE neutrophils exhibit distinct functional responses compared to control neutrophils. These functional differences may result from differential gene expression via miRNAs. Furthermore, the differences in functional phenotype of SLE neutrophils suggest that they may contribute to SLE differently dependent on the inflammatory milieu.

Genetic load in incomplete lupus erythematosus.

OBJECTIVE: Patients with incomplete lupus erythematosus (ILE) have lupus features but insufficient criteria for SLE classification. Some patients with ILE transition to SLE, but most avoid major organ involvement. This study tested whether the milder disease course in ILE is influenced by reduced SLE risk allele genetic load. METHODS: We calculated the genetic load based on 99 SLE-associated risk alleles in European American patients with SLE (≥4 American College of Rheumatology (ACR) 1997 criteria, n=170), patients with ILE (3 ACR 1997 criteria, n=169), a subset of patients with ILE not meeting Systemic Lupus International Collaborating Clinics (SLICC) classification (ILESLICC, n=119) and healthy controls (n=133). Unweighted genetic loads were calculated as the total sum of risk alleles for each individual, while weighted genetic loads were defined as the sum of risk alleles multiplied by the natural logarithm of the previously published OR of each risk allele for SLE susceptibility. RESULTS: The median unweighted and weighted SLE risk allele genetic load was significantly greater in patients with ILE (unweighted: 81, p value=0.01; weighted: 16.3, p value=0.001) and patients with SLE (80, p value=0.02; 16.29, p value=0.0006) compared with healthy controls (78, 15.76). Patients with ILESLICC trended towards an increased genetic load, although not statistically significant (unweighted: 80, p value=0.14; weighted: 16.05, p value=0.07). However, the median genetic load did not significantly differ between ILE and SLE, and genetic load did not differentiate patients with ILE and SLE (area under the curve=0.51, p=0.78) by receiver operator characteristic analysis. CONCLUSIONS: Patients with ILE and SLE have comparable genetic loads of SLE risk loci, suggesting similar genetic predispositions between these conditions. Phenotypical differences between SLE and ILE may instead be influenced by ILE-specific variants and gene-environment interactions.

A Flare Risk Index Informed by Select Immune Mediators in Systemic Lupus Erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is marked by immune dysregulation linked to varied clinical disease activity. Using a unique longitudinal cohort of SLE patients, this study sought to identify optimal immune mediators informing an empirically refined flare risk index (FRI) reflecting altered immunity prior to clinical disease flare. METHODS: Thirty-seven SLE-associated plasma mediators were evaluated by microfluidic immunoassay in 46 samples obtained in SLE patients with an imminent clinical disease flare (preflare) and 53 samples obtained in SLE patients without a flare over a corresponding period (pre-nonflare). SLE patients were selected from a unique longitudinal cohort of 106 patients with classified SLE (meeting the American College of Rheumatology 1997 revised criteria for SLE or the Systemic Lupus International Collaborating Clinics 2012 revised criteria for SLE). Autoantibody specificities, hybrid SLE Disease Activity Index (hSLEDAI) scores, clinical features, and medication usage were also compared at preflare (mean ± SD 111 ± 47 days prior to flare) versus pre-nonflare (99 ± 21 days prior to nonflare) time points. Variable importance was determined by random forest analysis with logistic regression subsequently applied to determine the optimal number and type of analytes informing a refined FRI. RESULTS: Preflare versus pre-nonflare differences were not associated with demographics, autoantibody specificities, hSLEDAI scores, clinical features, nor medication usage. Forward selection and backward elimination of mediators ranked by variable importance resulted in 17 plasma mediator candidates differentiating preflare from pre-nonflare visits. A final combination of 11 mediators best informed a newly refined FRI, which achieved a maximum sensitivity of 97% and maximum specificity of 98% after applying decision curve analysis to define low, medium, and high FRI scores. CONCLUSION: We verified altered immune mediators associated with imminent disease flare, and a subset of these mediators improved the FRI to identify SLE patients at risk of imminent flare. This molecularly informed, proactive management approach could be critical in prospective clinical trials and the clinical management of lupus.

Adults with systemic lupus exhibit distinct molecular phenotypes in a cross-sectional study.

BACKGROUND: The clinical and pathologic diversity of systemic lupus erythematosus (SLE) hinders diagnosis, management, and treatment development. This study addresses heterogeneity in SLE through comprehensive molecular phenotyping and machine learning clustering. METHODS: Adult SLE patients (n = 198) provided plasma, serum, and RNA. Disease activity was scored by modified SELENA-SLEDAI. Twenty-nine co-expression module scores were calculated from microarray gene-expression data. Plasma soluble mediators (n = 23) and autoantibodies (n = 13) were assessed by multiplex bead-based assays and ELISAs. Patient clusters were identified by machine learning combining K-means clustering and random forest analysis of co-expression module scores and soluble mediators. FINDINGS: SLEDAI scores correlated with interferon, plasma cell, and select cell cycle modules, and with circulating IFN-α, IP10, and IL-1α levels. Co-expression modules and soluble mediators differentiated seven clusters of SLE patients with unique molecular phenotypes. Inflammation and interferon modules were elevated in Clusters 1 (moderately) and 4 (strongly), with decreased T cell modules in Cluster 4. Monocyte, neutrophil, plasmablast, B cell, and T cell modules distinguished the remaining clusters. Active clinical features were similar across clusters. Clinical SLEDAI trended highest in Clusters 3 and 4, though Cluster 3 lacked strong interferon and inflammation signatures. Renal activity was more frequent in Cluster 4, and rare in Clusters 2, 5, and 7. Serology findings were lowest in Clusters 2 and 5. Musculoskeletal and mucocutaneous activity were common in all clusters. INTERPRETATION: Molecular profiles distinguish SLE subsets that are not apparent from clinical information. Prospective longitudinal studies of these profiles may help improve prognostic evaluation, clinical trial design, and precision medicine approaches. FUNDING: US National Institutes of Health.

Lupus patient decisions about clinical trial participation: a qualitative evaluation of perceptions, facilitators and barriers.

Objective: Although SLE disproportionately affects minority racial groups, they are significantly under-represented in clinical trials in the USA. This may lead to misleading conclusions in race-based subgroup analyses. We conducted focus groups to evaluate the perceptions of diverse patients with lupus about clinical trial participation. Methods: A qualitative research design employed three 90 min focus groups led by a trained moderator and guided by the Theory of Planned Behaviour. Open-ended questions about trial participation included advantages and disadvantages (behavioural beliefs), approving and disapproving significant others (normative beliefs), and participation enhancers and barriers (control beliefs). Discussions were recorded, transcribed and analysed to identify emerging themes. Results: Patients with SLE (n=23) aged 21-72, with increased proportion of minority groups (65%), participated. Reported advantages of trial participation included altruism and personal benefit. Disadvantages included uncertainties, disappointment, information burden, and life-health balance. Although some patients had discussed research participation with approving or disapproving family or friends, self-approval superseded external approval. Barriers included logistics and time, and facilitators included flexibility in scheduling, advance notice of studies, streamlined forms, and hope for SLE improvement. Conclusions: Knowledge about potential benefits of clinical trial participation was high. Minority patients demonstrated confidence in making their own informed decisions, but major barriers for all participants included burdensome forms, travel, childcare, and work. These suggest a major impact on minority and all recruitment from behavioural and control aspects, which should be considered in the logistics of trial design. This does not minimise the potential importance of improved access and education about clinical research.

Unique Sjögren's syndrome patient subsets defined by molecular features.

OBJECTIVE: To address heterogeneity complicating primary SS (pSS) clinical trials, research and care by characterizing and clustering patients by their molecular phenotypes. METHODS: pSS patients met American-European Consensus Group classification criteria and had at least one systemic manifestation and stimulated salivary flow of ⩾0.1 ml/min. Correlated transcriptional modules were derived from gene expression microarray data from blood (n = 47 with appropriate samples). Patients were clustered based on this molecular information using an unbiased random forest modelling approach. In addition, multiplex, bead-based assays and ELISAs were used to assess 30 serum cytokines, chemokines and soluble receptors. Eleven autoantibodies, including anti-Ro/SSA and anti-La/SSB, were measured by Bio-Rad Bioplex 2200. RESULTS: Transcriptional modules distinguished three clusters of pSS patients. Cluster 1 showed no significant elevation of IFN or inflammation modules. Cluster 2 showed strong IFN and inflammation modular network signatures, as well as high plasma protein levels of IP-10/CXCL10, MIG/CXCL9, BLyS (BAFF) and LIGHT. Cluster 3 samples exhibited moderately elevated IFN modules, but with suppressed inflammatory modules, increased IP-10/CXCL10 and B cell-attracting chemokine 1/CXCL13 and trends toward increased MIG/CXCL9, IL-1α, and IL-21. Anti-Ro/SSA and anti-La/SSB were present in all three clusters. CONCLUSION: Molecular profiles encompassing IFN, inflammation and other signatures can be used to separate patients with pSS into distinct clusters. In the future, such profiles may inform patient selection for clinical trials and guide treatment decisions.

Scoring systemic lupus erythematosus (SLE) disease activity with simple, rapid outcome measures.

OBJECTIVE: Existing methods for grading lupus flares or improvement require definition-based thresholds as increments of change. Visual analogue scales (VAS) allow rapid, continuous scaling of disease severity. We analysed the performance of the SELENA SLEDAI Physician's Global Assessment (SSPGA) and the Lupus Foundation of America-Rapid Evaluation of Activity in Lupus (LFA-REAL) as measures of improvement or worsening in SLE. METHODS: We evaluated the agreement between prospectively collected measures of lupus disease activity [SLE Disease Activity Index (SLEDAI), British Isles Lupus Assessment Group Index 2004 (BILAG 2004), Cutaneous Lupus Area and Severity Index (CLASI), SSPGA and LFA-REAL] and response [(SLE Responder Index (SRI)-4 and BILAG-Based Combined Lupus Assessment (BICLA)] in a clinical trial. RESULTS: Fifty patients (47 females, mean age 45 (±11.6) years) were assessed at 528 consecutive visits (average 10.6 (±4.1) visits/patient). Changes in disease activity compared with baseline were examined in 478 visit pairs. SSPGA and LFA-REAL correlated with each other (r=0.936), and with SLEDAI and BILAG (SSPGA: r=0.742 (SLEDAI), r=0.776 (BILAG); LFA-REAL: r=0.778 (SLEDAI), r=0.813 (BILAG); all p<0.0001). Changes (∆) in SSPGA and LFA-REAL compared with screening correlated with each other (r=0.857) and with changes in SLEDAI and BILAG (∆SSPGA: r=0.678 (∆SLEDAI), r=0.624 (∆BILAG); ∆LFA-REAL: r=0.686 (∆SLEDAI) and 0.700 (∆BILAG); all p<0.0001). Changes in SSPGA and LFA-REAL strongly correlated with SRI-4 and BICLA by receiver operating characteristic analysis (p<0.0001 for all). Additionally, LFA-REAL correlated to individual BILAG organ scores (musculoskeletal: r=0.842, mucocutaneous: r=0.826 (p<0.0001 for both)). CONCLUSION: SSPGA and LFA-REAL are reliable surrogates of common SLE trial end points and could be used as continuous or dichotomous response measures. Additionally, LFA-REAL can provide individualised scoring at the symptom or organ level. TRIAL REGISTRATION NUMBER: NCT02270957.

Comparison of the Lupus Foundation of America-Rapid Evaluation of Activity in Lupus to More Complex Disease Activity Instruments As Evaluated by Clinical Investigators or Real-World Clinicians.

OBJECTIVE: Lupus disease measures such as the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and the British Isles Lupus Assessment Group (BILAG) index are challenging to interpret. The Lupus Foundation of America-Rapid Evaluation of Activity in Lupus (LFA-REAL) is intended to provide an efficient application of anchored visual analog scores, each representing the individual severity of active symptoms, with the sum of individual scores deriving an overall disease activity assessment. Our objective was to compare the performance of LFA-REAL to systemic lupus erythematosus disease activity assessments and compare scores between trained lupus clinical investigators and clinicians. METHODS: Investigators scored the SLEDAI, BILAG, physician's global assessment (PGA), and LFA-REAL, while the clinicians scored the LFA-REAL. The level of agreement between physicians and instruments was determined. RESULTS: The study included 99 patients (93% women, 31% white, mean ± SD ages 43.4 ± 13.2 years). At the first visit, the mean ± SD SLEDAI score was 5.5 ± 4.5, BILAG score 6.7 ± 7.8, and PGA score 33.6 ± 24.5. The mean ± SD investigator LFA-REAL score was 46.2 ± 42.9, and clinician LFA-REAL score 56.1 ± 53.6. At the second visit, the mean ± SD investigator LFA-REAL score was 41.3 ± 36.7, and clinician LFA-REAL score 48.3 ± 42.6. Total LFA-REAL scores correlated positively with PGA, SLEDAI, and BILAG (ρ = 0.58-0.88, P < 0.001). LFA-REAL scores produced correlation coefficients of ρ > 0.7 for musculoskeletal, mucocutaneous, and renal BILAG domains. The intraclass correlation coefficient between the LFA-REAL scores of investigators and clinicians was 0.79 for visit 1 (P < 0.001) and 0.86 for visit 2 (P < 0.001). CONCLUSION: The LFA-REAL provides a reliable surrogate for more complicated disease activity measures when used by lupus clinical investigators or clinicians.

Use of SLICC criteria in a large, diverse lupus registry enables SLE classification of a subset of ACR-designated subjects with incomplete lupus.

OBJECTIVE: SLE is traditionally classified using the American College of Rheumatology (ACR) criteria. The Systemic Lupus International Collaborating Clinics (SLICC) recently validated an alternative system. This study examined large cohorts of subjects with SLE and incomplete lupus erythematosus (ILE) to compare the impact of ACR and SLICC criteria. METHODS: Medical records of subjects in the Lupus Family Registry and Repository were reviewed for documentation of 1997 ACR classification criteria, SLICC classification criteria and medication usage. Autoantibodies were assessed by indirect immunofluorescence (ANA, antidouble-stranded DNA), precipitin (Sm) and ELISA (anticardiolipin). Other relevant autoantibodies were detected by precipitin and with a bead-based multiplex assay. RESULTS: Of 3575 subjects classified with SLE under at least one system, 3312 (92.6%) were classified as SLE by both systems (SLEboth), 85 only by ACR criteria (SLEACR-only) and 178 only by SLICC criteria (SLESLICC-only). Of 440 subjects meeting 3 ACR criteria, 33.9% (149/440) were SLESLICC-only, while 66.1% (n=291, designated ILE) did not meet the SLICC classification criteria. Under the SLICC system, the complement criterion and the individual autoantibody criteria enabled SLE classification of SLESLICC-only subjects, while SLEACR-only subjects failed to meet SLICC classification due to the combined acute/subacute cutaneous criterion. The SLICC criteria classified more African-American subjects by the leucopenia/lymphopenia criterion than did ACR criteria. Compared with SLEACR-only subjects, SLESLICC-only subjects exhibited similar numbers of affected organ systems, rates of major organ system involvement (∼30%: pulmonary, cardiovascular, renal, neurological) and medication history. CONCLUSIONS: The SLICC criteria classify more subjects with SLE than ACR criteria; however, individuals with incomplete lupus still exist under SLICC criteria. Subjects who gain SLE classification through SLICC criteria exhibit heterogeneous disease, including potential major organ involvement. These results provide supportive evidence that SLICC criteria may be more inclusive of SLE subjects for clinical studies.

Clinical and Serologic Features in Patients With Incomplete Lupus Classification Versus Systemic Lupus Erythematosus Patients and Controls.

OBJECTIVE: Incomplete lupus erythematosus (ILE) involves clinical and/or serologic manifestations consistent with but insufficient for systemic lupus erythematosus (SLE) classification. Because the nature of ILE is poorly understood and no treatment recommendations exist, we examined the clinical manifestations, medication history, and immunologic features in a diverse collection of ILE and SLE patients. METHODS: Medical records of subjects enrolled in the Lupus Family Registry and Repository were reviewed for medication history and American College of Rheumatology (ACR) classification criteria to identify ILE patients (3 ACR criteria; n = 440) and SLE patients (≥4 ACR criteria; n = 3,397). Participants completed the Connective Tissue Disease Screening Questionnaire. Anticardiolipin and plasma B lymphocyte stimulator (BLyS) were measured by enzyme-linked immunosorbent assay, antinuclear antibodies (ANAs) by indirect immunofluorescence, and 13 autoantibodies by bead-based assays. RESULTS: On average, ILE patients were older than SLE patients (46.2 years versus 42.0 years; P < 0.0001), and fewer ILE patients were African American (23.9% versus 32.2%; P < 0.001). ILE patients exhibited fewer autoantibody specificities than SLE patients (1.3 versus 2.6; P < 0.0001) and were less likely to have ANA titers ≥1:1,080 (10.5% versus 19.5%; P < 0.0001). BLyS levels were intermediate in ILE patients (controls < ILE; P = 0.016; ILE < SLE; P = 0.008). Pericarditis, renal, or neurologic manifestations occurred in 12.5% of ILE patients and were associated with non-European American race/ethnicity (P = 0.012). Hydroxychloroquine use increased over time, but was less frequent in ILE than SLE patients (65.2% versus 83.1%; P < 0.0001). CONCLUSION: Although usually characterized by milder symptoms, ILE manifestations may require immunomodulatory treatments. Longitudinal studies are necessary to understand how ILE affects organ damage and future SLE risk, and to delineate molecular pathways unique to ILE.

Preliminary test of the LFA rapid evaluation of activity in lupus (LFA-REAL): an efficient outcome measure correlates with validated instruments.

OBJECTIVE: Current disease activity measures for systemic lupus erythematosus (SLE) are difficult to score or interpret and problematic for use in clinical practice. Lupus Foundation of America (LFA)-Rapid Evaluation of Activity in Lupus (REAL) is a pilot application composed of anchored visual analogue scores (0-100 mm each) for each organ affected by lupus. This study evaluated the use of LFA-REAL in capturing SLE disease activity. METHODS: In a preliminary test of LFA-REAL, this simplified, organ-based system was compared with the most widely used outcome measures in clinical trials, the British Isles Lupus Assessment Group 2004 Index (BILAG), the SLE Disease Activity Index (SLEDAI) and the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) SLEDAI Physician's Global Assessment (SS-PGA). The level of agreement was analysed using Spearman rank correlations. RESULTS: 91 patients with SLE with mild to severe disease activity were evaluated, their median SLEDAI score was 4.0 (range 0-28) and BILAG score 8.0 (0-32). The median SS-PGA was 38 mm (4-92) versus the total REAL 50 mm (0-268), which expands in range by additive organ scores. Thirty-three patients had moderate to severe disease activity (≥1.5 on SS-PGA landmarks). The median SS-PGA score of this group was 66 mm (50-92) versus median REAL score of 100 mm (59-268), confirming ability to detect a wider distribution of scores at higher disease activity. Total REAL correlated with SLEDAI, BILAG and SS-PGA (correlation coefficient=0.816, 0.933 and 0.903, respectively; p<0.001 for all). Individual LFA-REAL organ scores for musculoskeletal and mucocutaneous also correlated with corresponding BILAG domain scores (correlation coefficient=0.925 and 0.934, p<0.001). CONCLUSIONS: In this preliminary exercise, there were strong correlations between LFA-REAL and validated lupus disease activity indices. Further development may be valuable for consistent scoring in clinical trials, grading optimal assessment of change in disease activity and reliable monitoring of patients in practice.

Clarithromycin in adult-onset still's disease: a potentially useful therapeutic.

Adult-onset Still's disease (AOSD), an autoinflammatory syndrome of unknown etiology, typically manifests with spiking fevers, polyarthritis, and characteristic evanescent rash. We describe a young woman with AOSD complicated by calf fasciitis that serendipitously responded to clarithromycin administered for another indication. Remarkable improvement followed rechallenges with clarithromycin for subsequent AOSD flares. In addition to their antibacterial actions, macrolides demonstrate immunomodulatory effects, including suppression of proinflammatory cytokine production and neutrophil action. Previous clinical trials provide promising preliminary evidence of a therapeutic effect of macrolides in chronic inflammatory diseases. Although AOSD pathogenesis remains unclear, a role for dysregulation of innate immunity is supported by recent literature. Based on this possible innate immune mechanism, we suspect that macrolides may have induced a therapeutic response in this patient with AOSD. A clinical trial is warranted to establish or refute their therapeutic efficacy.

The lupus family registry and repository.

The Lupus Family Registry and Repository (LFRR) was established with the goal of assembling and distributing materials and data from families with one or more living members diagnosed with SLE, in order to address SLE genetics. In the present article, we describe the problems and solutions of the registry design and biometric data gathering; the protocols implemented to guarantee data quality and protection of participant privacy and consent; and the establishment of a local and international network of collaborators. At the same time, we illustrate how the LFRR has enabled progress in lupus genetics research, answering old scientific questions while laying out new challenges in the elucidation of the biologic mechanisms that underlie disease pathogenesis. Trained staff ascertain SLE cases, unaffected family members and population-based controls, proceeding in compliance with the relevant laws and standards; participant consent and privacy are central to the LFRR's effort. Data, DNA, serum, plasma, peripheral blood and transformed B-cell lines are collected and stored, and subject to strict quality control and safety measures. Coded data and materials derived from the registry are available for approved scientific users. The LFRR has contributed to the discovery of most of the 37 genetic associations now known to contribute to lupus through 104 publications. The LFRR contains 2618 lupus cases from 1954 pedigrees that are being studied by 76 approved users and their collaborators. The registry includes difficult to obtain populations, such as multiplex pedigrees, minority patients and affected males, and constitutes the largest collection of lupus pedigrees in the world. The LFRR is a useful resource for the discovery and characterization of genetic associations in SLE.

Autoantibody prevalence and lupus characteristics in a unique African American population.

OBJECTIVE: The Gullah population of the Sea Islands of South Carolina is a unique group of African Americans who, due to geographic and cultural factors, remained isolated with minimal genetic admixture until the 1950s. Because of the unique homogeneous nature of the Gullah, we sought to define the genetic and environmental factors contributing to systemic lupus erythematosus (SLE) in this population. METHODS: Using data from our ongoing cohort study of lupus in the Gullah population, which we established in 2003, disease characteristics and serologic profiles were summarized for 184 patients with SLE, 144 unaffected first-degree relatives, and 144 matched unrelated, unaffected control subjects. These findings were compared with those in 2 other large cohorts of African Americans with SLE. RESULTS: In the Gullah cohort, we observed a high prevalence of SLE multiplex families (26.6%), malar rash (56.0%), discoid rash (34.2%), photosensitivity (60.9%), and oral/nasal ulcerations (43.5%), but a lower prevalence of hematologic and pleuropericardial disease than has been reported in other African American cohorts. Overall renal and central nervous system involvement, number of American College of Rheumatology disease criteria met, and SLE Damage Index scores were similar to those reported in other cohorts. Of interest, male and female first-degree relatives and male and female control subjects in this cohort had similar rates of antinuclear antibody positivity, whereas lupus-specific antibodies were more prevalent in the women than in the men. CONCLUSION: These data indicate that the severity of lupus in the Gullah population is similar to that in other African American populations, whereas skin disease and familial disease prevalence are increased in the Gullah. These findings suggest that there is an increased genetic influence on overall disease in this cohort compared with that in other African American cohorts, which confirms the unique nature of this cohort.