Andrea Fava

Despite advances in lupus biomarker research, clinical translation has remained limited, leading to delayed diagnosis, imprecise disease monitoring and trial-and-error treatment approaches. This roadmap, developed by the Lupus Research Alliance’s Biomarker Roadmap Committee with input from patients and experts, identifies key priorities for translating research into clinical benefit. Three major clinical unmet needs are highlighted: improving diagnostic accuracy of SLE and neuropsychiatric SLE, developing sensitive longitudinal markers for disease activity (including LN), and predicting disease progression and treatment response. We review the current state of biomarker development across these domains and highlight barriers that hinder clinical validation, assay development and clinical adoption. We propose focused strategies to overcome these challenges, emphasizing analytically validated assays, diverse longitudinal cohorts, alignment with meaningful clinical outcomes and consortium-based efforts. The roadmap outlines a path to transform lupus diagnosis, monitoring and precision medicine, enabling research advances to improve patient care.

Neutrophils have been implicated in mediating immune crosstalk between injured tissue and distal organs, either promoting local and distal inflammation or mediating tissue repair. Skin injury from ultraviolet (UV) light triggers neutrophil migration to the kidney, which may have implications for nephritis flares in systemic lupus erythematosus. Here, we demonstrate that after skin exposure to UV light, renal neutrophils upregulate proangiogenic transcriptional programs and activate renal endothelial angiogenesis and tissue remodeling processes in healthy kidneys. Ligand-receptor analysis identified Cd177–Pecam1 as a key neutrophil-endothelial interaction. In contrast to healthy control mice, skin exposure to UV light stimulated the expression of Tgfβ and Wnt signaling pathways in lupus-prone MRL/lpr mice. This tissue remodeling response was neutrophil dependent. Spatial transcriptomic analysis of lupus nephritis kidneys revealed that tissue remodeling and profibrotic pathways are highly expressed in neutrophil-rich areas. Neutrophil intrarenal levels were higher in the kidneys of lupus nephritis patients with active skin disease, compared with those without skin involvement at the time of the nephritis flare. Moreover, urinary neutrophils were increased in systemic lupus erythematosus patients with active skin disease, including sun-associated skin rash, even in the absence of lupus nephritis. These results support a model in which UV-triggered skin injury activates a neutrophil-mediated skin-kidney axis that promotes differential tissue remodeling pathways in healthy compared with lupus-prone kidneys.

Lupus nephritis (LN), a severe manifestation of systemic lupus erythematosus (SLE), is a heterogeneous disease driven by diverse immune and tissue cell types. We obtained 538,194 single-cell and 142,881 single-nuclear profiles from kidney biopsies of 155 patients with LN and 30 preimplantation transplant biopsy controls, along with 327,326 single-cell blood profiles. We characterized key stromal and immune cell types and cell states; moreover, we distinguished cell states that were tissue specific from those that were also present in the blood. We observed that LN pathological features were associated with particular cell states. For example, after controlling for the effects of chronic tissue damage, we observed that expansion of glomerular and scar-associated macrophage populations correlated with increasing inflammatory disease activity. Scar-associated macrophages appear to drive LN fibrosis and, in active disease, infiltrate the glomeruli more than other myeloid cells. These observations support that therapeutic targeting of myeloid populations may offer a strategy to prevent renal inflammation and ongoing kidney damage in LN.

Background: Traditional immunohistochemistry (IHC) with chromogen detection has limited multiplex capacity, detecting at most 4 protein markers per tissue section simultaneously, thereby restricting comprehensive spatial analysis of valuable human biopsies. We developed and validated a robust serial IHC (sIHC) staining method to detect multiple antigens on a single kidney biopsy slide, maximizing data yield for diagnosing and studying complex kidney diseases. Methods: Formalin-fixed, paraffin-embedded kidney biopsy sections were subjected to repeated IHC/imaging cycles with antibody removal using an optimized sodium dodecyl sulfate-glycerol buffer stripping protocol. Images were then co-registered, and analysis was performed using a variety of methodologies, including color deconvolution, cell segmentation, and spatial clustering. Results: This optimized sIHC method successfully detected up to 20 antigens on a single slide. Combining image analysis and artificial intelligence software, for example with HALO (Indica Labs), the assay assembles high-dimensional images and enables quantitative histology and single-cell spatial analysis. Using this advanced method, we were able to identify rare cell populations, such as double-negative T cells, that are challenging to detect conventionally. Conclusion: We have developed a validated, high-capacity sIHC protocol that uses standard IHC procedures with commercially available, clinically validated off-the-shelf antibodies. This method is a valuable, cost-effective tool for obtaining extensive, high-dimensional single-cell-resolved spatial data from limited pathology samples, such as a human kidney biopsy.

Objectives: Active proliferative LN is associated with elevated urinary neutrophil granule proteins such as PR3, implicating active cellular degranulation in LN. Yet, mature neutrophils are rarely observed in LN biopsies, raising the possibility of an unrecognized cellular source. We aimed to identify and characterize PR3-expressing myeloid cells in LN kidneys using a spatially resolved multi-omics approach. Methods: Serial immunohistochemistry (10-plex) and spatial transcriptomics were employed to assess the distribution of PR3+ cells across renal tissue and characterize their cellular phenotype. Results: PR3+ cells were abundant in LN kidney biopsies, but with marked inter-patient heterogeneity. These cells were predominantly localized to the tubulointerstitium and showed similar distribution in both proliferative and membranous LN. In contrast, intraglomerular PR3+ cells were enriched in proliferative LN and correlated with the NIH activity index. Phenotype analysis identified four subsets of PR3+ cells, consistent with a monocyte/neutrophil lineage. Most PR3+ cells lacked multilobate nuclei, distinguishing them from mature neutrophils, suggesting that these cells may be overlooked by conventional light microscopy. Conclusions: PR3+ myeloid cells are a prominent and heterogeneous population in LN. While intraglomerular PR3+ cells were specific to proliferative LN and correlated with histologic activity, the widespread presence of PR3+ cells in the tubulointerstitium suggests broader involvement in renal inflammation than previously recognized. These findings reveal a potentially overlooked immune cell population that may contribute to renal injury in LN.

Importance: Cardiovascular disease (CVD) is a leading cause of morbidity and mortality in patients with Systemic Lupus Erythematosus (SLE), due to both traditional CVD risk factors and SLE specific factors. Although statins are first-line therapy for primary prevention of CVD in the general population, it is unclear whether statins protect against first time cardiovascular events (CVEs) in patients with SLE. Objective: Determine whether statins are protective in primary prevention of CVEs among patients with SLE. Design, setting, and participants: This cohort study is a retrospective analysis of a well-characterized, prospective cohort of patients with SLE with patient follow-up beginning in 2013. Main outcome and measures: CVEs were defined as the occurrence of myocardial infarction, thrombotic stroke, onset of angina, or coronary bypass procedure. Statin use in the prior year was quantified based on standardized defined daily doses (DDD). Rates of occurrence were compared using pooled logistic regression. A multivariable model was performed to adjust for possible confounders. Results: The analysis was based on 8708 person-years of follow-up from 1396 cohort participants: 1283 (92%) were women, 567 (41%) Black, and 665 (48%) White. Patients were stratified by use of statin within the last year: none, < standard DDD, or ≥ standard DDD. The rate of events per 1000 person-years was respectively 5.3, 8.5, and 8.0 (p=0.31) within these 3 groups, suggesting potential lack of protective effect of statin treatment. The rates of CVEs among statin versus non-statin users remained the same after adjusting for and stratifying by total cholesterol level (p=0.18). Significantly higher rates of CVEs occurred among those with BMI 25-30 kg/m ² (p=0.0066) and those prescribed ≥ 10 mg/day of prednisone (p=0.0003). Multivariable analysis also suggested a potential lack of protective effect of statins against CVEs (OR 1.48; 95% CI, 0.79-2.75; p=0.21883) and diabetes mellitus was found to be independently associated with an increased risk for development of CVEs (OR 4.48; 95% CI 1.99-10.08; p=0.00029). Conclusion and Relevance: Among patients with SLE, statin use may not be protective in primary prevention of CVEs, regardless of statin exposure. Prednisone use, history of diabetes mellitus, and elevated BMI were drivers of increased cardiovascular risk in univariate analysis. Diabetes mellitus persisted as an independent risk factor for CVEs in a multivariable model. Our work reinforces findings from clinical trials which have shown no reduction in subclinical measures of atherosclerosis with statin use among patients with SLE, as well as a mechanistic substudy which demonstrated that statins are ineffective in normalizing the pro-atherogenic changes induced by SLE.

Objective: Kidney survival is the ultimate goal in lupus nephritis (LN) management, but long‐term predictors remain inadequately studied, requiring long‐term follow‐up. This study aimed to identify baseline and early longitudinal predictors of kidney survival in the Accelerating Medicines Partnership LN longitudinal cohort. Methods: We performed time‐to‐event analyses of clinical, centrally scored histologic, and serological predictors of kidney function loss (sustained ≥40% estimated glomerular filtration rate [eGFR] decline or progression to end‐stage kidney disease) in 172 LN patients with a median follow‐up of 4.6 years (range 0.5–7.8). Results: Kidney function loss occurred in 57 of 172 (33%) patients. Baseline lower eGFR, non‐first kidney biopsy, and higher National Institutes of Health (NIH) chronicity index were associated with eGFR loss. Chronicity index was the strongest predictor, but no clear threshold defined higher risk. NIH activity index and International Society of Nephrology (ISN) class were not predictive. Proteinuria at 12 months was prognostic, with urine protein‐to‐creatinine ratio <0.7 g/g associated with lower risk; however, no single threshold ensured protection, and lower levels had better outcomes. Lack of complete clinical response at 3, 6, or 12 months predicted future eGFR loss, whereas partial response conferred intermediate risk. Serological markers were not associated with eGFR loss. Conclusion: Low baseline eGFR and chronic histologic damage, but not activity or ISN class, predicted eGFR loss. Proteinuria <0.7 g/g at one year was associated with better outcomes but did not ensure protection. Because proteinuria does not reflect intrarenal inflammation, these results suggest current response definitions serve better as prognostic indicators than true measures of treatment efficacy, and better biomarkers are needed. image

Lupus nephritis (LN), a severe manifestation of systemic lupus erythematosus (SLE), features heterogeneous renal pathology and reliance on invasive biopsies for diagnosis, prognosis, and treatment selection. Current peripheral clinical markers inadequately capture disease activity and progression. Here, we performed comprehensive serum proteomic profiling of over 5,000 proteins in the large, longitudinal Accelerating Medicines Partnership Rheumatoid Arthritis/SLE cohort of 270 LN patients and 63 healthy controls. Machine learning identified distinct molecular signatures that classified LN versus controls, differentiated histological classes, and delineated activity- and chronicity-associated pathways, including inflammatory cytokine, PI3K/AKT, TGFb;, and complement/coagulation pathways. An increase in VSIG4, CD27, HAVCR1, and LAIR1 consistently emerged as top biomarkers across multiple clinical contexts, and early decreases in these markers at 3 months were associated with complete treatment response at 1 year. By resolving coordinated serum protein modules linked to key inflammatory, PI3K/AKT, TGFb;, and complement pathways, these signatures mechanistically connect circulating proteomic perturbations to intrarenal immune activation, tissue injury, and repair in LN. These findings demonstrate that serum proteomics reflect complex intrarenal immunopathology and offer a promising noninvasive liquid biopsy approach to refine LN classification and guide personalized management, potentially reducing the need for repeated invasive biopsies and improving therapeutic decision-making.

Objective: Lupus nephritis (LN) is one of the most severe manifestations of systemic lupus erythematosus (SLE) and is partially driven by type I interferon signaling. Anifrolumab, an approved treatment for patients with SLE, has been investigated in a phase 2 trial in patients with LN receiving standard therapy (TULIP‐LN, ClinicalTrials.gov identifier NCT02547922). We studied the impact of anifrolumab treatment on urinary biomarker expression in patients with LN through proteomic analysis of samples from TULIP‐LN. Methods: Urine samples were collected at weeks 0, 12, and 48 from patients treated with the anifrolumab basic regimen (n = 35), intensified regimen (n = 42), or placebo (n = 35), in addition to standard therapy, and analyzed for the presence of 197 proteins. The impact of anifrolumab relative to placebo on prespecified biomarkers linked to histologic activity was assessed, and a comparison of responders versus nonresponders was conducted on proteins detected in ≥75% of samples. Results: Anifrolumab treatment significantly reduced urinary CD163 and monocyte chemoattractant protein 1 expression at week 12 versus placebo, including in patients classified as proteinuric nonresponders across all regimens. By week 48, biomarker levels declined in all groups, indicating that standard therapy alone can eventually suppress intrarenal inflammation but with slower kinetics. Proteomic analyses further revealed that anifrolumab was superior to placebo in reducing the proteomic inflammatory signature, regardless of responder status. Conclusion: Compared with placebo, anifrolumab treatment significantly reduced urinary biomarkers of renal histologic activity in patients with LN, which may accelerate the resolution of intrarenal inflammation, potentially preventing damage accrual. image

Objective: We evaluated the role of 25‐hydroxyvitamin D (25[OH]D), prednisone, and other risk factors for bone mineral density (BMD) loss and osteoporosis in systemic lupus erythematosus (SLE). Methods: We calculated the association between 25(OH)D levels and other potential risk factors and BMD measures (spine T scores) and osteoporosis (defined as a T score ≤ −2.5) in a lupus cohort of 1,003 patients. Generalized estimating equations were used to assess the statistical significance of observed differences, accounting for patients with multiple BMD measures. Results: Univariate analysis showed that older age, lower body mass index (BMI), mean past SLE activity (Safety of Estrogens in Lupus Erythematosus National Assessment–Systemic Lupus Erythematosus Disease Activity Index > 2), mean daily prednisone dose at any level, and a history of low C3 or C4 were associated with osteoporosis. Older age, lower BMI, mean 25(OH)D levels, mean daily prednisone dose (again at any dose), and prior low C3 were associated with lower spine T scores. In the multivariate analysis for osteoporosis, older age, lower BMI, low mean 25(OH)D levels, and prednisone dose >5 mg/day remained predictors. Discontinuation of prednisone within one year before the next BMD measurement led to higher mean spine T scores. Conclusion: Prednisone dose, even at 5 mg/day, and low mean 25(OH)D levels were found to be modifiable predictors of osteoporosis. Mean (reflecting supplementation), rather than initial, 25(OH)D levels were significantly associated with BMD. Some of the bone loss from prior prednisone use appears to be reversible.