Interferon and B-cell Signatures Inform Precision Medicine in Lupus Nephritis

Introduction Current therapeutic management of lupus nephritis (LN) fails to induce long-term remission in over 50% of patients, highlighting the urgent need for additional options. Methods We analyzed differentially expressed genes (DEGs) in peripheral blood from patients with active LN (n = 41) and active nonrenal lupus (n = 62) versus healthy controls (HCs) (n = 497) from the European PRECISESADS project (NTC02890121), and dysregulated gene modules in a discovery (n = 26) and a replication (n = 15) set of active LN cases. Results Replicated gene modules qualified for correlation analyses with serologic markers, and regulatory network and druggability analysis. Unsupervised coexpression network analysis revealed 20 dysregulated gene modules and stratified the active LN population into 3 distinct subgroups. These subgroups were characterized by low, intermediate, and high interferon (IFN) signatures, with differential dysregulation of the “B cell” and “plasma cells/Ig” modules. Drugs annotated to the IFN network included CC-motif chemokine receptor 1 (CCR1) inhibitors, programmed death-ligand 1 (PD-L1) inhibitors, and irinotecan; whereas the anti-CD38 daratumumab and proteasome inhibitor bortezomib showed potential for counteracting the “plasma cells/Ig” signature. In silico analysis demonstrated the low-IFN subgroup to benefit from calcineurin inhibition and the intermediate-IFN subgroup from B-cell targeted therapies. High-IFN patients exhibited greater anticipated response to anifrolumab whereas daratumumab appeared beneficial to the intermediate-IFN and high-IFN subgroups. Conclusion IFN upregulation and B and plasma cell gene dysregulation patterns revealed 3 subgroups of LN, which may not necessarily represent distinct disease phenotypes but rather phases of the inflammatory processes during a renal flare, providing a conceptual framework for precision medicine in LN.


For all patients For all patients
Age ≥18 years Diagnosed according to the prevailing criteria for systemic lupus erythematosus (SLE) Informed consent signed Pediatric lupus Drug-induced lupus Severe nephrotic syndrome with proteinuria ≥3.5 g/day Patients with stable doses of prednisone equivalent >15 mg/day for the last 3 months or with IV corticosteroids in the last 3 months Patients under immunosuppressant treatment in the last 3 months prior to recruitment and patients with combined therapy using two or more immunosuppressants: • Methotrexate ≥25mg/week • Azathioprine ≥2.5mg/kg/day • Cyclosporine A >3mg/kg/day • Mycophenolate mofetil >2g/day Chronic HBV or HCV infection Patients who are also diagnosed according to the prevailing criteria for one of the following autoimmune diseases: • Rheumatoid arthritis (RA) • Scleroderma or systemic sclerosis (SSc) • Primary Sjögren's syndrome (pSjS) • Primary antiphospholipid syndrome (pAPS) • Mixed connective tissue disease (MCTD) • Patients with undifferentiated connective tissue disease (UCTD) for over 1 year and that do not fulfill the diagnosis of any of the above diseases or SLE

For controls
Individuals on chronic medication Individuals suffering from any inflammatory autoimmune, allergic or infectious condition, and with a history of autoimmune disease, particularly thyroid disease or other diseases that may modify cellular profiles in blood

Supplementary Table S3. Renal disease activity in LN subgroups Target lo-IFN subgroup im-IFN subgroup hi-IFN subgroup lo-IFN vs im-IFN lo-IFN vs hi-IFN im-IFN vs hi-IFN N=13 N=9 N=19 p value p value p value
Adapted from "Integrative Analysis Reveals a Molecular Stratification of Systemic Autoimmune Diseases."Arthritis Rheum.2021;73(6):1073-1085.Copyright 2021 by the American College of Rheumatology.Adapted with permission.IV: intravenous, HBV: hepatitis B virus, HCV: hepatitis C virus.

Supplementary Table S4. Correlations between serological markers and z-scores of the "B cell" gene module in patients with LN Serological marker n Coefficient p value anti-MDA IgM
Spearman's rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the B cell gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Correlations between serological markers and z-scores of the "cell cycle, mitotic phase" gene module in patients with LN
Spearman's rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the cell cycle, mitotic phase gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Correlations between serological markers and z-scores of the "CORO1A-DEF6 network" gene module in patients with LN
Spearman's rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the CORO1A-DEF6 network gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Correlations between serological markers and z-scores of the "enriched in antigen presentation (III)" gene module in patients with LN
Supplementary TableS8.

Correlations between serological markers and z-scores of the "enriched in neutrophils (II)" gene module in patients with LN
rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the enriched in neutrophils (II) gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Correlations between serological markers and z-scores of the "erythrocytes" gene module in patients with LN
rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the erythrocytes gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Correlations between serological markers and z-scores of the "extracellular matrix (I)" gene module in patients with LN
rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the extracellular matrix (I) gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Correlations between serological markers and z-scores of the "extracellular region cluster" gene module in patients with LN
Supplementary TableS12.

Correlations between serological markers and z-scores of the "inflammation (IV)" gene module in patients with LN
Supplementary TableS13.

Correlations between serological markers and z-scores of the "inflammation (VI)" gene module in patients with LN
Supplementary TableS14.

Correlations between serological markers and z-scores of the "inositol phosphate metabolism" gene module in patients with LN
rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the inositol phosphate metabolism gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Correlations between serological markers and z-scores of the "interferon" gene module in patients with LN
Supplementary TableS16.

Correlations between serological markers and z-scores of the "interferon (II)" gene module in patients with LN
rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the interferon (II) gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Correlations between serological markers and z-scores of the "MAPK, RAS signalling" gene module in patients with LN
rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the MAPK, RAS signalling gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Table S19 . Correlations between serological markers and z-scores of the "plasma cells, immunoglobulins" gene module in patients with LN
rank correlation coefficients of correlations between levels of different serological markers and Z-scores of the plasma cells, immunoglobulins gene module.The total number of patients with available data is indicated.Statistically significant p values are in bold.Only comparisons with sufficient numbers of observations (n≥10) are included.

Table S20 . Correlations between serological markers and z-scores of the "platelets" gene module in patients with LN
Supplementary TableS22.

Correlations between serological markers and z-scores of the "regulation of antigen presentation and immune response" gene module in patients with LN
Supplementary TableS23.

Correlations between serological markers and z-scores of the "regulation of transcription, transcription factors" gene module in patients with LN
Supplementary TableS33.

of antigen presentation and immune response gene module
Genes in replicated gene modules with a mean |z-score| >1 in at least one LN patient subgroup were imputed in iRegulon through Cytoscape to generate signalling molecule networks and identify their chief regulators.The top chief regulators and enriched motifs are displayed.AUC: area under the curve; LN: lupus nephritis; NES: normalised enrichment score; TFs: transcription factors.