Identification of a neutrophil-related gene expression signature that is enriched in adult systemic lupus erythematosus patients with active nephritis: Clinical/pathologic associations and etiologic mechanisms

Abstract

Both a lack of biomarkers and relatively ineffective treatments constitute impediments to management of lupus nephritis (LN). Here we used gene expression microarrays to contrast the transcriptomic profiles of active SLE patients with and without LN to identify potential biomarkers for this condition. RNA isolated from whole peripheral blood of active SLE patients was used for transcriptomic profiling and the data analyzed by linear modeling, with corrections for multiple testing. Results were validated in a second cohort of SLE patients, using NanoString technology. The majority of genes demonstrating altered transcript abundance between patients with and without LN were neutrophil-related. Findings in the validation cohort confirmed this observation and showed that levels of RNA abundance in renal remission were similar to active patients without LN. In secondary analyses, RNA abundance correlated with disease activity, hematuria and proteinuria, but not renal biopsy changes. As abundance levels of the individual transcripts correlated strongly with each other, a composite neutrophil score was generated by summing all levels before examining additional correlations. There was a modest correlation between the neutrophil score and the blood neutrophil count, which was largely driven by the dose of glucocorticosteroids and not the proportion of low density and/or activated neutrophils. Analysis of longitudinal data revealed no correlation between baseline neutrophil score or changes over the first year of follow-up with subsequent renal flare or treatment outcomes, respectively. The findings argue that although the neutrophil score is associated with LN, its clinical utility as a biomarker may be limited.

Fig 1. Differences in RNA abundance between patient groups.

(A) Transcriptomic profiles for the 171 genes with the highest abundance variance, as determined by microarray, in the whole blood of 38 SLE patients and 17 controls are shown. Normalized signal intensities were adjusted using row-wise mean centering with SD scaling, with blue indicating over-expression and green indicating under-expression. Hierarchical clustering was performed on the samples (row) and genes (columns) using divisive analysis. (B) Log₂ fold-change of normalized RNA abundance for comparisons between patient groups (ALN, active lupus nephritis; ANLN, active no lupus nephritis; RLN, remission lupus nephritis), as determined by NanoString. Top covariates indicate the experimental (purple) and control (green) groups being compared in each column. The size of the circles indicates the magnitude of the fold-change for each comparison, with orange indicating over-expression and blue indicating under-expression in the experimental group. The background in each cell indicates the statistical significance of the comparison as determined by multivariate linear modeling followed by FDR correction. Results for 11 IFN-induced genes are shown at the top of the figure and are separated from 15 genes examined that were identified as differentially expressed between renal and non-renal disease in the microarray study, which are shown at the bottom of the figure. (C) Results for representative neutrophil-expressed genes, showing the similarity between gene expression profiles in the discovery and validation cohorts. (D) Correlation of normalized RNA abundance with clinical and laboratory variables. Data is expressed similarly to that shown in panel B, except that the clinical variables assessed are shown at the bottom of the figure. Statistical significance was determined by Spearman correlation followed by FDR correction of p-values.

Fig 2. Association between the neutrophil score, GCS dose, and neutrophil count in the SLE patients.

(A) Neutrophil scores in healthy controls (HC) and SLE patients stratified by disease group; active lupus without (ANLN) and with (ALN) nephritis, as well as lupus nephritis in remission (RLN). The dashed line represents 3 SD above the mean for HC. Significant differences from healthy controls are indicated by asterisks (*** p < 0.001, **** p < 0.0001), with the p values for significant differences between groups shown above the bars. (B) Association between GCS dose and the neutrophil score for all SLE patients. (C) Neutrophil scores stratified based upon GCS dose (shown at the bottom of the figure) with 3 groups <10 mg, 10–20 mg, and >20 mg (all GCS doses have been converted to their prednisone equivalent). Significant differences between the levels in ALN patients and other patient groups are shown (* p < 0.05, ** p < 0.01). (D) Neutrophil counts in SLE patients stratified by disease group. Significant differences between groups (p values) are shown above the bars. (E) Association between neutrophil count and the neutrophil score for all SLE patients. (F) Neutrophil counts stratified based upon GCS dose (shown at the bottom of the figure) with 3 groups <10 mg, 10–20 mg, and >20 mg (all GCS doses have been converted to their prednisone equivalent). Significant differences between the levels in ALN patients and other patient groups are shown (** p < 0.01).

Fig 3. Correlation between the neutrophil score, LDGs, and neutrophil activation.

(A) Flow plots showing the strategy for gating LDGs. PBMCs were isolated over a Ficoll gradient and then the CD10⁺CD15⁺ granulocytes gated as shown. As shown in the panels on the right, these cells were CD14^loCD16⁺ and had a unique forward (FSC) and side (SSC) scatter profile, consistent with the reported LDG phenotype [24]. (B) Correlation between the neutrophil score and the number of LDGs per ml or neutrophil count. (C) Flow plot showing the region used to gate activated (CD11b^hiCD66b^hi) cells within the whole peripheral blood CD10⁺CD15⁺ neutrophil population. (D) Correlation between the neutrophil score, the number of activated neutrophils or LDGs in the peripheral blood.

Fig 4. Correlation between neutrophil and clinical parameters over time.

(A) Representative results showing the neutrophil score during longitudinal follow-up for five independent patients with LN. Scales on the left indicate neutrophil score, whereas those on the right give the values for the clinical parameters examined. The GCS doses were divided by 10 in order to enable them to be expressed on the same scale as the SLEDAI-2K and neutrophil counts. Numbers in the top right corner indicate the ISN renal biopsy class. (B) Comparison of changes in the neutrophil score over time in patients stratified by clinical response at 2 years following initiation of treatment for biopsy proven LN.